Name: fluorimetric techniques for the assessment of sperm membranes
Uploaded: 2018-11-28T13:00:00
Description: Watch this Scientific Journal Video about Fluorimetric Techniques for the Assessment of Sperm Membranes at JoVE.com

The authors would like to thank &#34;SION&#34; Israeli company for artificial insemination and breeding (Hafetz-Haim, Israel) for their help and cooperation, and Ms. Li Na (IMV Technologies, L&#39;Aigle, France) for assistance with the instrument setup and training.

All of the experiments were performed in accordance with the 1994 Israeli guidelines for animal welfare. Bovine sperm was supplied by commercial Israeli company for artificial insemination and breeding. Ejaculates of 11 bulls were evaluated in this study.
1. Sperm Sample Preparation
NOTE: The procedure is based on the Roth laboratory&#39;s protocol1,3.
<ol>
	<li>Obtain approximately 1&#821...

<ol>
	<li>Komsky-Elbaz, A., Roth, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+the+herbicide+atrazine+and+its+metabolite+DACT+on+bovine+sperm+quality.">Effect of the herbicide atrazine and its metabolite DACT on bovine sperm quality.</a> Reprod Toxicol. 67, 15-25 (2016).</li><li>G&#252;rler, H., 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=Effects+of+cryopreservation+on+sperm+viability,+synthesis+of+reactive+oxygen+species,+and+DNA+damage+of+bovine+sperm.">Effects of cryopreservation on sperm viability, synthesis of reactive oxygen species, and DNA damage of bovine sperm.</a> Theriogenology. 86 (2), 562-571 (2016).</li><li>Komsky-Elbaz, A., Saktsier, M., Roth, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aflatoxin+B1+impairs+sperm+quality+and+fertilization+competence.">Aflatoxin B1 impairs sperm quality and fertilization competence.</a> Toxicology. 393, 42-50 (2018).</li><li>Beltr&#225;n, C., 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=Role+of+Ion+Channels+in+the+Sperm+Acrosome+Reaction.">Role of Ion Channels in the Sperm Acrosome Reaction.</a> Adv Anat Embryol Cell Biol. 220, 35-69 (2016).</li><li>Breitbart, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+pathways+in+sperm+capacitation+and+acrosome+reaction.">Signaling pathways in sperm capacitation and acrosome reaction.</a> Cell Mol Biol (Noisy-le-grand). 49 (3), 321-327 (2003).</li><li>Almadaly, E., 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=Methodological+factors+affecting+the+results+of+staining+frozen-thawed+fertile+and+subfertile+Japanese+Black+bull+spermatozoa+for+acrosomal+status.">Methodological factors affecting the results of staining frozen-thawed fertile and subfertile Japanese Black bull spermatozoa for acrosomal status.</a> Anim Reprod Sci. 136 (1-2), 23-32 (2012).</li><li>Jankovicov&#225;, J., Simon, M., Antal&#237;kov&#225;, J., Horovsk&#225;, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acrosomal+and+viability+status+of+bovine+spermatozoa+evaluated+by+two+staining+methods.">Acrosomal and viability status of bovine spermatozoa evaluated by two staining methods.</a> Vet Hung. 56 (1), 133-138 (2008).</li><li>Lybaert, P., Danguy, A., Leleux, F., Meuris, S., Lebrun, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+methodology+for+the+detection+and+quantification+of+the+acrosome+reaction+in+mouse+spermatozoa.">Improved methodology for the detection and quantification of the acrosome reaction in mouse spermatozoa.</a> Histol Histopathol. 24 (8), 999-1007 (2009).</li><li>Whitfield, C. H., Parkinson, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+fertility+of+bovine+semen+and+in+vitro+induction+of+acrosome+reactions+by+heparin.">Relationship between fertility of bovine semen and in vitro induction of acrosome reactions by heparin.</a> Theriogenology. 38 (1), 11-20 (1992).</li><li>Celeghini, E. C. C., de Arruda, R. P., de Andrade, A. F. C., Nascimento, J., Raphael, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Practical+Techniques+for+Bovine+Sperm+Simultaneous+Fluorimetric+Assessment+of+Plasma,+Acrosomal+and+Mitochondrial+Membranes.">Practical Techniques for Bovine Sperm Simultaneous Fluorimetric Assessment of Plasma, Acrosomal and Mitochondrial Membranes.</a> Reprod Domest Anim. 42 (5), 479-488 (2007).</li><li>Ramalho-Santos, J., Varum, S., Amaral, S., Mota, P. C., Sousa, A. P., Amaral, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+functionality+in+reproduction:+from+gonads+and+gametes+to+embryos+and+embryonic+stem+cells.">Mitochondrial functionality in reproduction: from gonads and gametes to embryos and embryonic stem cells.</a> Hum Reprod. 15 (5), 553-572 (2009).</li><li>Eddy, E. M., O&#8217;Brien, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The spermatozoon. , (1994).</li><li>Gallon, F., Marchetti, C., Jouy, N., Marchetti, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+functionality+of+mitochondria+differentiates+human+spermatozoa+with+high+and+low+fertilizing+capability.">The functionality of mitochondria differentiates human spermatozoa with high and low fertilizing capability.</a> Fertil Steril. 86 (5), 1526-1530 (2006).</li><li>Espinoza, J. a., Paasch, U., Villegas, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+membrane+potential+disruption+pattern+in+human+sperm.">Mitochondrial membrane potential disruption pattern in human sperm.</a> Hum Reprod. 24 (9), 2079-2085 (2009).</li><li>H&#252;ttemann, M., Lee, I., Pecinova, A., Pecina, P., Przyklenk, K., Doan, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+oxidative+phosphorylation,+the+mitochondrial+membrane+potential,+and+their+role+in+human+disease.">Regulation of oxidative phosphorylation, the mitochondrial membrane potential, and their role in human disease.</a> J Bioenerg Biomembr. 40 (5), 445-456 (2008).</li><li>Sellem, E., 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=Use+of+combinations+of+in+vitro+quality+assessments+to+predict+fertility+of+bovine+semen.">Use of combinations of in vitro quality assessments to predict fertility of bovine semen.</a> Theriogenology. 84 (9), 1447-1454 (2015).</li><li>Odhiambo, J. F., Sutovsky, M., DeJarnette, J. M., Marshall, C., Sutovsky, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+of+ubiquitin-PNA+based+sperm+quality+assay+for+semen+evaluation+by+a+conventional+flow+cytometer+and+a+dedicated+platform+for+flow+cytometric+semen+analysis.">Adaptation of ubiquitin-PNA based sperm quality assay for semen evaluation by a conventional flow cytometer and a dedicated platform for flow cytometric semen analysis.</a> Theriogenology. 76 (6), 1168-1176 (2011).</li><li>Barrier Battut, I., Kempfer, A., Becker, J., Lebailly, L., Camugli, S., Chevrier, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+new+fertility+prediction+model+for+stallion+semen,+including+flow+cytometry.">Development of a new fertility prediction model for stallion semen, including flow cytometry.</a> Theriogenology. 86 (4), 1111-1131 (2016).</li></ol>

Sperm fertilization potential depends on multiple factors reflecting its quality. A high concentration of spermatozoa and a high proportion of highly progressively motile spermatozoa might be considered high-quality semen. Nevertheless, such an evaluation does not take into account other cellular and functional parameters. The use of &#39;bench-top&#39; microcapillary flow cytometer can be easily adapted to evaluation of various sperm structures using fluorescent probes, as previously shown by others17<...

Integrity and functionality of sperm membranes are a few of the factors indicating sperm viability and fertilization potential. The plasma membrane acts as a barrier between intracellular and extracellular compartments, thereby maintaining the cellular osmotic equilibrium1. Any stress that induces damage to the plasma membrane integrity might impair homeostasis, reduce viability and fertilization capacity, and increase cell death. For instance, cryopreservation reduces sperm viability due to damage to its plasma membrane, as a result of temperature changes and osmotic stress2. We previously reported that exposing bull sperm to low concentrations of foodborne contaminants such as the pesticide atrazine, its major metabolite diaminochlorotriazine or the mycotoxin aflatoxin B1, reduces sperm viability1,3. This was determined by labeling the double-stranded DNA with DAPI in combination with PI, which binds to the DNA of cells with a damaged plasma membrane.
Acrosome reaction (AR) involves fusion&#160;of the outer acrosome membrane and the overlying plasma membrane resulting in the release of acrosomal enzymes4,5. These are essential events for zona-pellucida penetration and further merging of the sperm with the oocyte6. Therefore, evaluation of acrosomal membrane integrity constitutes a useful parameter to evaluate the semen quality and male fertility7,8,9. Several fluorescent techniques are suitable for the verification of acrosome integrity, FITC-PNA or FITC-PSA8,10. In our previous studies, using the patterns of FITC-PSA staining1,3, we provided accurate definitions for (i) intact acrosome, (ii) damaged acrosome membrane and (iii) reacted acrosome. In the current report, we evaluate acrosome status using sperm-dedicated flow cytometry and compare the results to those using fluorescence microscopy.
The mitochondria are multifunctional organelles involved in, among other things, ATP synthesis, reactive oxygen species production, calcium signaling and apoptosis. Physiological dysfunctions, including male and female infertility, are associated with altered mitochondrial function11.Sperm mitochondria are arranged in the midpiece and play a crucial role in sperm motility12. It is well accepted that high mitochondrial membrane potential (&#916;&#936;m) is associated with normal motility and high fertilization capacity13. In contrast, low &#916;&#936;m&#160;is associated with an elevated level of reactive oxygen species and reduced fertilization rate14. Nonetheless, various environmental compounds, for instance endocrine disruptors, can induce cellular stress and lead to a transient increase in &#916;&#936;m, hyperpolarization1,3, increased production of free radicals and eventually, apoptosis15. The fluorescent probe 5,5&#39;,6,6&#39;-tetra-chloro-1,1&#39;,3,3&#39;-tetraethylbenzimidazolyl carbocyanine iodide (JC-1) enables examining for example, the effects of foodborne toxins on sperm &#916;&#936;m1,3.
Standard spermiograms, based on physiological and morphological parameters, are not good enough to predict semen quality. More accurate methods are required to ensure sperm quality. Here, we provide two feasible methods to determine sperm quality based on evaluations of sperm membranes: simultaneous quadruple staining with specific fluorescent probes and fluorescence microscopy, described in our studies1,3 and advanced sperm-dedicated flow cytometry, recently utilized in our laboratory, and already being used by others16,17,18.

<table>
	<tbody>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S5886</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P5405</td>
			<td></td>
		</tr>
		<tr>
			<td>MOPS [3-N-morphilino propanesulfonic acid]</td>
			<td>Sigma</td>
			<td>M1254</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma</td>
			<td>P5493</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D2438</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>Sigma</td>
			<td>64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Neubauer Germany</td>
			<td></td>
			<td>hemocytometer</td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-diamidino-2-phenylindole)</td>
			<td>Sigma</td>
			<td>D9542</td>
			<td>fluorescent probe</td>
		</tr>
		<tr>
			<td>PI (propidium iodide )</td>
			<td>Sigma</td>
			<td>P4170</td>
			<td>fluorescent probe</td>
		</tr>
		<tr>
			<td>FITC-PSA (fluorescein isothiocyanate-conjugated Pisum sativum agglutinin )</td>
			<td>Sigma</td>
			<td>L0770</td>
			<td>fluorescent probe</td>
		</tr>
		<tr>
			<td>JC-1 (5,5&#39;,6,6&#39;-tetra-chloro-1,1&#39;,3,3&#39;-tetraethylbenzimidazolyl carbocyanine iodide)</td>
			<td>ENZOBiochem, New York, NY, USA</td>
			<td>ENZ52304</td>
			<td>fluorescent probe</td>
		</tr>
		<tr>
			<td>Annexin V conjugated to FITC</td>
			<td>MACS, Miltenyi Biotec</td>
			<td>130-093-060</td>
			<td>fluorescent probe</td>
		</tr>
		<tr>
			<td>Annexin V binding buffer 20X stock solution</td>
			<td>MACS, Miltenyi Biotec</td>
			<td>130-092-820</td>
			<td>buffer</td>
		</tr>
		<tr>
			<td>Nikon Eclipse, TE-2000-u</td>
			<td>Nikon, Tokyo, Japan</td>
			<td></td>
			<td>inverted fluorescence microscope</td>
		</tr>
		<tr>
			<td>Nis Elements</td>
			<td>Nikon, Tokyo, Japan</td>
			<td></td>
			<td>software</td>
		</tr>
		<tr>
			<td>Nikon DXM1200F</td>
			<td>Nikon, Tokyo, Japan</td>
			<td></td>
			<td>digital camera</td>
		</tr>
		<tr>
			<td>Guava EasyCyte Plus</td>
			<td>IMV Technologies, L&#39;Aigle, France</td>
			<td></td>
			<td>microcapillary sperm flow cytometer</td>
		</tr>
		<tr>
			<td>CytoSoft</td>
			<td>Guava Technologies Inc., Hayward, CA, USA; distributed by IMV Technologies</td>
			<td></td>
			<td>software</td>
		</tr>
		<tr>
			<td>Buffered solution for cytometry</td>
			<td>IMV Technologies, L&#39;Aigle, France</td>
			<td>023862</td>
			<td>buffer</td>
		</tr>
		<tr>
			<td>Viability and concentration kit</td>
			<td>IMV Technologies, L&#39;Aigle, France</td>
			<td>024708</td>
			<td>kit for viability assessment</td>
		</tr>
		<tr>
			<td>Mitochondrial activity kit</td>
			<td>IMV Technologies, L&#39;Aigle, France</td>
			<td>024864</td>
			<td>kit for mitochondrial activity assessment</td>
		</tr>
		<tr>
			<td>Viability &#38; acrosome integrity kit</td>
			<td>IMV Technologies, L&#39;Aigle, France</td>
			<td>025293</td>
			<td>kit for acrosome integrity assessment</td>
		</tr>
		<tr>
			<td>JMP-13</td>
			<td>SAS Institute Inc., 2004, ary, NC, USA</td>
			<td></td>
			<td>software</td>
		</tr>
		<tr>
			<td>Bovine sperm</td>
			<td>&#34;SION&#34;, Israeli company for artificial insemination and dreeding, Hafetz-Haim, Israel</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

fluorimetric techniques for the assessment of sperm membranes

Standard spermiograms describing sperm quality are mostly based on the physiological and visual parameters, such as ejaculate volume and concentration, motility and progressive motility, and sperm morphology and viability. However, none of these assessments is good enough to predict the semen quality. Given that maintenance of sperm viability and fertilization potential depends on membrane integrity and intracellular functionality, evaluation of these parameters might enable a better prediction of sperm fertilization competence. Here, we describe three feasible methods to evaluate sperm quality using specific fluorescent probes combined with fluorescence microscopy or flow cytometry analyses. Analyses assessed plasma membrane integrity using 4&#39;,6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI), acrosomal membrane integrity using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and mitochondrial membrane integrity using 5,5&#39;,6,6&#39;-tetra-chloro-1,1&#39;,3,3&#39;-tetraethylbenzimidazolyl carbocyanine iodide (JC-1). Combinations of these methods are also presented. For instance, use of annexin V combined with PI fluorochromes enables assessing apoptosis and calculating the proportion of apoptotic sperm (apoptotic index). We believe that these methodologies, which are based on examining spermatozoon membranes, are very useful for the evaluation of sperm quality.

Figure 1 shows simultaneous fluorimetric assessment of sperm membranes (plasma, acrosomal and mitochondrial) using PI, DAPI, FITC-PSA and JC-1. Assessment of sperm membranes using simultaneous staining with four fluorescent probes allows, for example, evaluating the proportion of sperm in each category&#8212;live vs. dead; high vs. low &#916;&#936;m; intact vs. damaged acrosome&#8212;simultaneously for each spermatozoon.
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Watch this Scientific Journal Video about Fluorimetric Techniques for the Assessment of Sperm Membranes at JoVE.com

Fluorimetric Techniques for the Assessment of Sperm Membranes

Here, we present methodologies to evaluate spermatozoan membrane integrity, a cellular feature associated with sperm fertilization competence. We describe three techniques for the fluorimetric assessment of sperm membranes: simultaneous staining with specific fluorescent probes, fluorescence microscopy, and advanced sperm-dedicated flow cytometry. Examples of combining the methodologies are also presented.

Standard spermiograms describing sperm quality are mostly based on the physiological and visual parameters, such as ejaculate volume and concentration, ...

The goal of these procedures is to evaluate sperm membranes integrity using specific fluorescent probes combined with fluorescence microscopy or flow cytometry analysis. These techniques might help answer key questions in the field of reproduction such as prediction of fertilization potential and quality of sperm sample. The main advantage of these techniques is that they enable precise evaluation of multiple sperm membranes which are known to associate with sperm fertilization potential.The implications of these techniques extend toward diagnosis of ejaculate quality as it enables more accurate evaluation of sperm plasma and acrosome membrane integrity as well as mitochondrial membrane potential. To begin this procedure, obtain approximately one to six milliliters of bull semen in a 15 milliliter tube at room temperature. Add six milliliters of NKM buffer pre-warmed to 37 degrees Celsius to each one milliliter of semen.Centrifuge at 600 times g for eight minutes. Repeat the centrifugation once or twice until the supernatant is clear. After this, immediately discard most of the supernatant, leaving approximately one centimeter of supernatant above the pellet.Carefully lean the tubes at a 30 degree angle in the incubator and wait 20 to 30 minutes to allow the spermatozoa to swim up. Using a micropipette, carefully remove the remaining one milliliter of supernatant which now contains the motile spermatozoa and transfer it to a fresh 1.5 milliliter tube. Keep the sperm at 37 degrees Celsius until ready to use.First, prepare all needed stock solutions as outlined in the text protocol. Next, transfer 133 microliters of the motile spermatozoa at a concentration of 25 million sperms per milliliter to a new 1.5 milliliter tube. Add 17 microliters of the DAPI working solution and incubate at 37 degrees Celsius for 10 minutes.Centrifuge at 1, 000 times g for five minutes. Discard the supernatant and add 100 microliters of NKM buffer to the pellet. Next, add 50 microliters of FITC-PSA, two microliters of JC-1, and three microliters of PI.Incubate at 37 degrees Celsius for 10 minutes.Centrifuge at 1, 000 times g for five minutes. Discard the supernatant and resuspend the pellet in 40 microliters of NKM buffer. Then transfer 10 microliters of the sample to a glass slide.Smear the sample and add a coverslip. After this, immediately begin visualizing the sample by epifluorescence microscopy with a 40X objective and a triple filter as outlined in the text protocol, using a digital camera to capture a separate image for each filter. Use the merge option in the camera software to merge the three images received from the filters, saving the new file in JPEG format.Open the merged image with the paint tool and use the brush option to mark the counted spermatozoa. Next, classify the spermatozoa based on the fluorescence emitted from each probe, making sure to evaluate at least 200 spermatozoa per slide. All cells should appear blue from the DAPI.Count the dead cells, which appear purple, to evaluate the viability. Then use the patterns of fluorescence staining to evaluate the acrosome status. Lastly, evaluate the mitochondrial membrane potential by distinguishing the spermatozoa with high mitochondrial membrane potential which exhibit a red-stained midpiece from the spermatozoa with low mitochondrial membrane potential which exhibit a green-stained midpiece.Count red and green midpieces separately and calculate their ratio. To begin the plasma membrane integrity evaluation, take the desired number of wells from the viability and concentration kit. Transfer the wells to the working base and cover them with a flexible lid to protect them from light.Add 199 microliters of buffered solution for cytometry to each well. Then add one microliter of homogenous semen at a concentration of 57 million cells per milliliter to each well and homogenize by pipetting. Cover the plate with the lid and incubate at 37 degrees Celsius for 10 minutes.After this, run the sample through the flow cytometer using the viability setting. To begin assessing the mitochondrial membrane potential, take out the desired number of wells from the mitochondrial activity kit. Transfer them to the working base.Add 10 microliters of absolute ethanol to each well and use a pipette to resuspend the powder present within the well. Add 190 microliters of PBS per well and homogenize by pipetting. Add 0.75 microliters of homogenous semen at a concentration of 57 million cells per milliliter to each well and homogenize by pipetting.Cover the plate with the lid and incubate at 37 degrees Celsius for 30 minutes. After this, run the sample through the flow cytometer using the mitochondrial activity setting. To begin assessing the acrosomal membrane integrity, take out the desired number of wells from the viability and acrosome integrity kit.Transfer the wells to the working base and cover them with a flexible lid to protect them from light. Add 200 microliters of buffered solution for cytometry to each well. Then add 0.7 microliters of homogenous semen at a concentration of 57 million cells per milliliter to each well and homogenize by pipetting.Cover the plate with the lid and incubate at 37 degrees Celsius for 45 minutes. After this, run the sample through the flow cytometer using the insight setting. In this study, semen quality is evaluated using different techniques, one of which evaluated sperm membranes using fluorometric probes.It is possible to evaluate the differences in sperm sample quality in terms of membrane integrity. For example, the ejaculate of bull number seven had a relatively low percentage of dead cells, a low proportion of sperm with pseudo-reacted acrosome, and higher mitochondrial membrane potential when compared to the ejaculate of bull number one. Samples are then evaluated for viability and mitochondrial activity.The histograms for these representative examples represent the ungated spermatozoa and debris and the gated spermatozoa. These histograms also represent the viable and dead cells and the distribution of spermatozoa to polarized and de-polarized mitochondrial membrane. The acrosome integrity is then evaluated with a ready-to-use kit and read with flow cytometry, dividing the area into three marker areas that represent negligible low fluorescing cells with intact unstained acrosome, low fluorescing cells with residual stained part of the acrosome, and highly fluorescing cells with disrupted acrosome.While attempting these procedures, it is important to make sure to prepare properly the initial sperm sample. These techniques can provide insight into sperm quality evaluation and can be applied to various organisms such as bovine, poultry, and human. Comparison between the two techniques, quadruple staining and flow cytometry indicated no significant differences for viability, mitochondrial membrane potential, and the acrosome integrity revealing that the two techniques produced matching results.

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Developmental Biology

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the field of environmental health. This is due in part to the paucity of model systems that can (1) accurately recapitulate keys features of reproductive processes and (2) do so in a medium- to high-throughput fashion, without the need for a high number of vertebrate animals.
We describe here an assay in the nematode C. elegans that allows the rapid identification of germline toxicants by monitoring the induction of aneuploid embryos. By making use of a GFP reporter line, errors in chromosome segregation resulting from germline disruption are easily visualized and quantified by automated fluorescence microscopy. Thus the screening of a particular set of compounds for its toxicity can be performed in a 96- to 384-well plate format in a matter of days. Secondary analysis of positive hits can be performed to determine whether the chromosome abnormalities originated from meiotic disruption or from early embryonic chromosome segregation errors. Altogether, this assay represents a fast first-pass strategy for the rapid assessment of germline dysfunction following chemical exposure.

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

We describe the detailed steps of a high-throughput chemical assay in the nematode Caenorhabditis elegans used to assess germline toxicity. In this assay, disruption of germline function following chemical exposure is monitored using a fluorescent reporter specific to aneuploid embryos.

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the ...

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under- or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development.

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during ...

Assessment of Maternal Vascular Remodeling During Pregnancy in the Mouse Uterus

The placenta mediates the exchange of factors such as gases and nutrients between mother and fetus and has specific demands for supply of blood from the maternal circulation. The maternal uterine vasculature needs to adapt to this temporary demand and the success of this arterial remodeling process has implications for fetal growth. Cells of the maternal immune system, especially natural killer (NK) cells, play a critical role in this process. Here we describe a method to assess the degree of remodeling of maternal spiral arteries during mouse pregnancy. Hematoxylin and eosin-stained tissue sections are scanned and the size of the vessels analysed. As a complementary validation method, we also present a qualitative assessment for the success of the remodeling process by immunohistochemical detection of smooth muscle actin (SMA), which normally disappears from within the arterial vascular media at mid-gestation. Together, these methods enable determination of an important parameter of the pregnancy phenotype. These results can be combined with other endpoints of mouse pregnancy to provide insight into the mechanisms underlying pregnancy-related complications.

This protocol describes a technique to assess changes in the maternal vasculature during pregnancy in mice. Using stereological methods, remodeling of the decidual spiral arteries is assessed quantitatively and the results confirmed qualitatively using immunohistochemistry.

The placenta mediates the exchange of factors such as gases and nutrients between mother and fetus and has specific demands for supply of blood from the ...

Assessment of the Effects of Endocrine Disrupting Compounds on the Development of Vertebrate Neural Network Function Using Multi-electrode Arrays

Bis-phenols, such as bis-phenol A (BPA) and bis-phenol-S (BPS), are polymerizing agents widely used in the production of plastics and numerous everyday products. They are classified as endocrine disrupting compounds (EDC) with estradiol-like properties. Long-term exposure to EDCs, even at low doses, has been linked with various health defects including cancer, behavioral disorders, and infertility, with greater vulnerability during early developmental periods. To study the effects of BPA on the development of neuronal function, we used an in vitro neuronal network derived from the early chick embryonic brain as a model. We found that exposure to BPA affected the development of network activity, specifically spiking activity and synchronization. A change in network activity is the crucial link between the molecular target of a drug or compound and its effect on behavioral outcome. Multi-electrode arrays are increasingly becoming useful tools to study the effects of drugs on network activity in vitro. There are several systems available in the market and, although there are variations in the number of electrodes, the type and quality of the electrode array and the analysis software, the basic underlying principles, and the data obtained is the same across the different systems. Although currently limited to analysis of two-dimensional in vitro cultures, these MEA systems are being improved to enable in vivo network activity in brain slices. Here, we provide a detailed protocol for embryonic exposure and recording neuronal network activity and synchrony, along with representative results.

Exposure to environmental toxins can acutely impact developing embryos. Endocrine disrupting chemicals such as bisphenols are known to adversely affect the nervous system. Here we describe a protocol using an in vitro vertebrate (chick embryo) neuronal network model to study the functional impact of toxin exposure on early embryos.

Bis-phenols, such as bis-phenol A (BPA) and bis-phenol-S (BPS), are polymerizing agents widely used in the production of plastics and numerous everyday ...

Handling and Treatment of Male European Eels (Anguilla anguilla) for Hormonal Maturation and Sperm Cryopreservation

During the last years, several research groups have been working on the development and improvement of new protocols for the European eel handling and maturation. As of yet, weekly injections of human chorionic gonadotropin (hCG) have proved to maturate males after just 5-6 weeks of treatment, producing high volumes of high-quality sperm during several weeks. In addition, sperm cryopreservation protocols using different extenders, cryoprotectants and cooling and thawing times have been previously described for European eel. Here, we show that Tanaka&#180;s extender solution can be directly used for fertilization or for cryopreservation, making unnecessary the usage of different types of solutions and dilutions. Furthermore, the use of methanol as a cryoprotectant makes this protocol easy to use as methanol has low toxicity and does not activate the sperm. The sperm does not need to be cryopreserved immediately after the addition of the cryoprotectant, and it can be used long after being thawed. Moreover, sperm motility is still high after thawing although it is lower than that of fresh sperm. The aim of this work is to show the best available protocol for European eel handling, maturation, and sperm cryopreservation.

Handling and Treatment of Male European Eels Anguilla anguilla for Hormonal Maturation and Sperm Cryopreservation

Protocols for European eel maturation and sperm cryopreservation have been improved over the last years. This article describes the best protocol available using human chorionic gonadotropin (hCG) for inducing maturation and methanol as cryoprotectant.

During the last years, several research groups have been working on the development and improvement of new protocols for the European eel handling and ...

Microfluidic Assay for the Assessment of Leukocyte Adhesion to Human Induced Pluripotent Stem Cell-derived Endothelial Cells (hiPSC-ECs)

Endothelial cells (ECs) are essential for the regulation of inflammatory responses by either limiting or facilitating leukocyte recruitment into affected tissues via a well-characterized cascade of pro-adhesive receptors which are upregulated on the leukocyte cell surface upon the inflammatory trigger. Inflammatory responses differ between individuals in the population and the genetic background can contribute to these differences. Human induced pluripotent stem cells (hiPSCs) have been shown to be a reliable source of ECs (hiPSC-ECs), thus representing an unlimited source of cells that capture the genetic identity and any genetic variants or mutations of the donor. hiPSC-ECs can therefore be used for modeling inflammatory responses in donor-specific cells. Inflammatory responses can be modeled by determining leukocyte adhesion to the hiPSC-ECs under physiological flow. This step-by-step protocol provides a detailed description of the experimental setup and data analysis for the assessment of inflammatory responses in hiPSC-ECs and the analysis of leukocyte adhesion under physiological flow.

Microfluidic Assay for the Assessment of Leukocyte Adhesion to Human Induced Pluripotent Stem Cell-derived Endothelial Cells hiPSC-ECs

This step-by-step protocol provides a detailed description of the experimental setup and data analysis for the assessment of inflammatory responses in hiPSC-ECs and the analysis of leukocyte adhesion under physiological flow.

Endothelial cells (ECs) are essential for the regulation of inflammatory responses by either limiting or facilitating leukocyte recruitment into affected ...

Assessment of Ultrastructural Neuroplasticity Parameters After In Utero Transduction of the Developing Mouse Brain and Spinal Cord

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ultrastructural parameters in unambiguously identified topographical structures, affected by a protein of interest that is introduced into the organism via viral transfer. This combined approach allows for a smooth transition from macrostructural to ultrastructural identification by following topographical navigation maps in a tissue atlas. High-resolution electron microscopy of the in-utero-transduced tissue reveals the fine ultrastructure of the neuropil and its plasticity parameters, such as cross-sectioned synaptic bouton areas, the number of synaptic vesicles and mitochondria within a bouton profile, the length of synaptic contacts, cross-sectioned axonal areas, the thickness of myelin sheaths, the number of myelin lamellae, and cross-sectioned areas of mitochondria profiles. The analysis of these parameters reveals essential insights into changes of ultrastructural plasticity in the areas of the nervous system that are affected by the viral transfer of the genetic construct. This combined method can not only be used for studying the direct effect of genetically engineered biomolecules and/or drugs on neuronal plasticity but also opens the possibility to study the in utero rescue of neuronal plasticity (e.g., in the context of neurodegenerative diseases).

The combination of transmission electron microscopy and in utero transduction is a powerful approach for studying morphological changes in the fine ultrastructure of the nervous system during development. This combined method allows deep insights into changes in structural details underlying neuroplasticity with respect to their topographical representation.

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ...

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

Successful fertilization is fundamental to sexual reproduction, yet little is known about the mechanisms that guide sperm to oocytes within the female reproductive tract. While in vitro studies suggest that sperm of internally fertilizing animals can respond to various cues from their surroundings, the inability to visualize their behavior inside the female reproductive tract creates a challenge for understanding sperm migration and mobility in its native environment. Here, we describe a method using C. elegans that overcomes this limitation and takes advantage of their transparent epidermis. C. elegans males stained with a mitochondrial&#160; dye are mated with adult hermaphrodites, which act as modified females, and deposit fluorescently labeled sperm into the hermaphrodite uterus. The migration and motility of the labeled sperm can then be directly tracked using an epi-fluorescence microscope in a live hermaphrodite. In wild-type animals, approximately 90% of the labeled sperm crawl through the uterus and reach the fertilization site, or spermatheca. Images of the uterus can be taken 1 h after mating to assess the distribution of the sperm within the uterus and the percentage of sperm that have reached the spermatheca. Alternatively, time-lapse images can be taken immediately after mating to assess sperm speed, directional velocity and reversal frequency. This method can be combined with other genetic and molecular tools available for the C.elegans to identify novel genetic and molecular mechanisms that are important in regulating sperm guidance and motility within the female reproductive tract.

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

Sperm must successfully navigate through the oviduct to fertilize an oocyte. Here, we describe an assay for measuring sperm migration within the C. elegans hermaphrodite uterus. This assay can provide quantitative data on sperm distribution within the uterus after mating, as well as on speed, directional velocity, and reversal frequency.

Successful fertilization is fundamental to sexual reproduction, yet little is known about the mechanisms that guide sperm to oocytes within the female ...

Evaluation of Fertilization State by Tracing Sperm Nuclear Morphology in Arabidopsis Double Fertilization

Flowering plants have a unique sexual reproduction system called &#8216;double fertilization&#39;,&#160;in which each of the sperm cells precisely fuses with an egg cell or a central cell. Thus, two independent fertilization events take place almost simultaneously. The fertilized egg cell and central cell develop into zygote and endosperm, respectively. Therefore, precise control of double fertilization is essential for the ensuing seed development. Double fertilization occurs in the female gametophyte (embryo sac), which is deeply hidden and covered with thick ovule and ovary tissues. This pistil tissue construction makes observation and analysis of double fertilization quite difficult and has created the present situation in which many questions regarding the mechanism of double fertilization remain unanswered. For the functional evaluation of a potential candidate for fertilization regulator, phenotypic analysis of fertilization is important. To judge the completion of fertilization in Arabidopsis thaliana, the shapes of fluorescence signals labeling sperm nuclei are used as indicators. A sperm cell that fails to fertilize is indicated by a condensed fluorescence signal outside of the female gametes, whereas a sperm cell that successfully fertilizes is indicated by a decondensed signal due to karyogamy with the female gametes&#8217; nucleus. The method described here provides a tool to determine successful or failed fertilization under in vivo conditions.

Evaluation of Fertilization State by Tracing Sperm Nuclear Morphology in Arabidopsis Double Fertilization

We demonstrate a method to determine successful or failed fertilization on the basis of sperm nuclear morphology in Arabidopsis double fertilization using an epifluorescence microscope.

Flowering plants have a unique sexual reproduction system called &#8216;double fertilization&#39;,&#160;in which each of the sperm cells precisely fuses ...

Application of Mouse Parthenogenetic Haploid Embryonic Stem Cells as a Substitute of Sperm

In organisms with sexual reproduction, germ cells are the source of totipotent cells that develop into new individuals. In mice, fertilization of an oocyte by a spermatozoon creates a totipotent zygote. Recently, several publications have reported that haploid embryonic stem cells (haESCs) can be a substitute for gametic genomes and contribute to embryos, which develop into mice. Here, we present a protocol to apply parthenogenetic haESCs as a substitute of sperm to construct embryos by intracytoplasmic injection into oocytes. This protocol consists of steps for preparing haESCs as sperm replacement, for injection of haESC chromosomes into oocytes, and for culture of semi-cloned embryos. The embryos can yield fertile semi-cloned mice after embryo transfer. Using haESCs as sperm replacement facilitates genome editing in the germline, studies of embryonic development, and investigation of genomic imprinting.

This article aims to demonstrate the use of parthenogenetic haploid embryonic stem cells as a substitute for sperm for the construction of semi-cloned embryos.

In organisms with sexual reproduction, germ cells are the source of totipotent cells that develop into new individuals. In mice, fertilization of an ...