This work is supported by College of Veterinary Medicine (WSU) start-up fund to WW. The authors thank Sierra Olsen (WSU) for critical reading of this manuscript.

All animals and procedures used in this study were handled according to Washington State University (WSU) Animal Care and Use Committee guidelines and in compliance with WSU-approved animal protocols #4702 and #4735.
1. Mice
<ol>
	<li>Use standard C57BL/6J adult male mice or other strains of interest. Maintain animals under a temperature- and humidity-controlled environment, with ad libitum access of water and food.
	<ol>
		<li>Use male breeders ranging from 8 weeks old to 10 months old. Use only proven breeders in the experiments.</li>
	</ol>
	</li>
	<li>Use adult female mice with a tissue selective deletion of reproductive tract estrogen receptor &#945; (encoded by Esr1 gene) in the epithelial cells10 (Wnt7aCre/+;Esr1f/f, called knockout or &#34;KO&#34;) and control littermates (Esr1f/f, called &#34;CTRL&#34;) in this study.
	<ol>
		<li>Use 8 week-old females. Ages of females varied depending on the experiment of interest.</li>
	</ol>
	</li>
	<li>Determine the stages of the estrous cycle in the morning at 08:00h. Use only females at the estrus stage of the cycle. For detailed methods of vaginal smearing and cytological analysis, refer to the previously published procedures11.</li>
</ol>
2. Mating and Assessing Copulatory Plugs
<ol>
	<li>At the day of estrus (16:00h), house the female in a male&#39;s cage.</li>
	<li>The next morning (08:00h), separate the female from the male mouse.</li>
	<li>Use an adaptation of a previously published method12 to access the presence or absence of a vaginal or copulatory plug as an indication of mating.</li>
	<li>Hold the female mouse from the base of the tail to briefly lift the hind limbs.</li>
	<li>Find the copulatory plug by the presence of an off-white seminal coagulum at the vaginal opening using a toothpick. If the copulatory plug is present, the toothpick will not be able to be inserted into the vaginal canal.</li>
</ol>
3. Preparation Before Tissue Collection
<ol>
	<li>Prepare 10 mL of Leibovitz&#39;s L-15 media, supplemented with 1% fetal bovine serum (called L15 media + 1% FBS) in a 15 mL tube. Incubate the media at 37 &#176;C for 15 min in a waterbath.</li>
	<li>In order to preserve the viability of the tissue and sperm for downstream applications, aliquot 2 mL of pre-warmed L15 media + 1% FBS in a 2 mL microcentrifuge tube for tissue collection.</li>
	<li>Warm the stage of the stereomicroscope to 37 &#176;C. Set the heat-block to 37 &#176;C and place an empty 1.5 mL microcentrifuge tube in the block.</li>
	<li>If the downstream applications require imaging of sperm motility, warm the glass slide to 37 &#176;C.</li>
</ol>
4. Tissue Collection
<ol>
	<li>Bring pre-warmed L15 media + 1% FBS aliquot to the tissue collection area.</li>
	<li>Euthanize the female (~08:30h) using carbon dioxide asphyxiation followed by cervical dislocation. 
	NOTE: This is approximately 8 h after mating, assuming that mating takes place at midnight.</li>
	<li>Place the animal in a supine position to expose the abdominal area.</li>
	<li>Spray 70% alcohol on the abdomen near hind limbs.</li>
	<li>Use the dissecting scissors to make a small cut (~1 cm) on the skin of the lower abdomen.</li>
	<li>Use the dominant hand to pull the tail (and hind limbs if necessary) while non-dominant hand pulls the skin in the opposite direction (toward the head) to expose the abdominal muscle. This method is used to minimize the amount of hair contamination to the visceral organs.</li>
	<li>Cut the abdominal muscle in a V-shape at the base of the abdomen, adjacent to the vagina, to expose the visceral organs.</li>
	<li>Move intestines and abdominal fat to the side to expose the reproductive tract.</li>
	<li>Trim off bladder and other tissues above the vaginal area.</li>
	<li>Cut pubic symphysis apart to gain access to the vaginal tissue.</li>
	<li>Lift the vaginal tissue at the vaginal opening and using scissors to separate the vaginal canal from the rectal tissue.</li>
	<li>Carefully trim off the mesenteric fat from both sides of the uterus using spring scissors. 
	CAUTION: The uterus is very fragile with the semen inside. Be careful not to puncture the uterus, or else the semen will be lost.</li>
	<li>Lift the lower reproductive tract and cut the ovarian fat pad off of both sides.</li>
	<li>Transfer the whole reproductive tract into the tube of pre-warmed Leibovitz&#39;s L-15 media + 1% FBS.</li>
</ol>
5. Measurement of Semen Liquefaction (Viscosity) from Uterine Content Collection
<ol>
	<li>On the heated stage of the stereomicroscope, transfer the tissues into a 35 mm sterile tissue culture dish filled with 3.5 mL pre-warmed L-15 media + 1% FBS to clean the tissues of blood and other contaminants. 
	CAUTION: While washing, grab the tissues by the fat, not the uterus. At this point, the semen is expected be intact inside the uterus.</li>
	<li>Blot the tissues of excess media using laboratory wipes.</li>
	<li>Hold the tissue at the vaginal end while placing the ovarian ends in the pre-heated microcentrifuge tube. While holding the vaginal end, cut the uterine horns at the base of the uterus, allowing the uterine horns to fall into the microcentrifuge tube.</li>
	<li>Let the semen flow out of the uterine horns into the tube. Use toothless forceps to gently squeeze the uterine horns from the ovarian end to the vaginal end, expelling out leftover semen.</li>
	<li>Discard the tissues in biohazard bag and incinerate.</li>
	<li>Briefly spin down the semen in a minicentrifuge (~1-2 s).</li>
	<li>Lay the tube, parallel to the heated stage - semen will not spill out.</li>
	<li>Have the timer ready and set to &#39;count up&#39;.</li>
	<li>Insert a 25 &#181;L capillary tube into the semen sample at a 180&#176; angle (parallel to the tube wall). This is to minimize the variability between each investigator from holding the capillary tube at different angles.</li>
	<li>Press the timer as soon as the capillary tube makes contact with the semen.</li>
	<li>Record the time (s) it takes for the semen to reach the 25 &#181;L line.</li>
	<li>When the measurement is finished, immediately put the tube containing semen back in the dry heat block for further functional analysis, such as video imaging of sperm motility.</li>
	<li>Disinfect the capillary tube containing semen by immersing the tube in 10% bleach solution for 20 min and discard the capillary tube in the sharps container.</li>
</ol>
6. Video Imaging of Sperm Motility
<ol>
	<li>Turn on the microscope and video imaging software.</li>
	<li>Pipette 20 &#181;L of the remaining semen onto the pre-warmed glass slide, while the glass slide is placed on the heated stage.</li>
	<li>Cover with a glass coverslip.</li>
	<li>Place the slide coverslip side down on the microscope.</li>
	<li>Use 20X objective lens to find area of interest.</li>
	<li>Change to 100X objective lens for video imaging.</li>
	<li>Record the video at 12 frames/s (fps) for 30 s.</li>
	<li>Randomly record the videos in at least 3 different areas per animal.</li>
	<li>Perform the imaging quickly and minimize light exposure to the slide in order to preserve sperm motility and viability. 
	NOTE: This imaging procedure should be done within 2-3 min.</li>
	<li>Perform the data analysis using ImageJ software as previously described8.</li>
</ol>

<ol>
	<li>Suarez, S. S., Pacey, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sperm+transport+in+the+female+reproductive+tract.">Sperm transport in the female reproductive tract.</a> Hum Reprod Update. 12 (1), 23-37 (2006).</li><li>Bedford, J. M., Yanagimachi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+of+sperm+motility+after+mating+in+the+rat+and+hamster.">Initiation of sperm motility after mating in the rat and hamster.</a> J Androl. 13 (5), 444-449 (1992).</li><li>Lilja, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+biology+of+semenogelin.">Cell biology of semenogelin.</a> Andrologia. 22 Suppl 1, 132-141 (1990).</li><li>Lilja, H., Abrahamsson, P. A., Lundwall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Semenogelin,+the+predominant+protein+in+human+semen.+Primary+structure+and+identification+of+closely+related+proteins+in+the+male+accessory+sex+glands+and+on+the+spermatozoa.">Semenogelin, the predominant protein in human semen. Primary structure and identification of closely related proteins in the male accessory sex glands and on the spermatozoa.</a> J Biol Chem. 264 (3), 1894-1900 (1989).</li><li>Veveris-Lowe, T. L., Kruger, S. J., Walsh, T., Gardiner, R. A., Clements, 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=Seminal+fluid+characterization+for+male+fertility+and+prostate+cancer:+kallikrein-related+serine+proteases+and+whole+proteome+approaches.">Seminal fluid characterization for male fertility and prostate cancer: kallikrein-related serine proteases and whole proteome approaches.</a> Semin Thromb Hemost. (33), 87-99 (2007).</li><li>Emami, N., Deperthes, D., Malm, J., Diamandis, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Major+role+of+human+KLK14+in+seminal+clot+liquefaction.">Major role of human KLK14 in seminal clot liquefaction.</a> J Biol Chem. 283 (28), 19561-19569 (2008).</li><li>Gerhard, I., Roth, B., Eggert-Kruse, W., Runnebaum, B. <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+kallikrein+on+sperm+motility,+capillary+tube+test,+and+pregnancy+rate+in+an+AIH+program.">Effects of kallikrein on sperm motility, capillary tube test, and pregnancy rate in an AIH program.</a> Arch Androl. 24 (2), 129-145 (1990).</li><li>Li, S., Garcia, M., Gewiss, R. L., Winuthayanon, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crucial+role+of+estrogen+for+the+mammalian+female+in+regulating+semen+coagulation+and+liquefaction+in+vivo.">Crucial role of estrogen for the mammalian female in regulating semen coagulation and liquefaction in vivo.</a> PLoS Genet. 13 (4), e1006743 (2017).</li><li>Elia, J., 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=Human+semen+hyperviscosity:+prevalence,+pathogenesis+and+therapeutic+aspects.">Human semen hyperviscosity: prevalence, pathogenesis and therapeutic aspects.</a> Asian J Androl. 11 (5), 609-615 (2009).</li><li>Winuthayanon, W., Hewitt, S. C., Orvis, G. D., Behringer, R. R., Korach, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uterine+epithelial+estrogen+receptor+alpha+is+dispensable+for+proliferation+but+essential+for+complete+biological+and+biochemical+responses.">Uterine epithelial estrogen receptor alpha is dispensable for proliferation but essential for complete biological and biochemical responses.</a> Proc Natl Acad Sci U S A. 107 (45), 19272-19277 (2010).</li><li>McLean, A. C., Valenzuela, N., Fai, S., Bennett, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performing+vaginal+lavage,+crystal+violet+staining,+and+vaginal+cytological+evaluation+for+mouse+estrous+cycle+staging+identification.">Performing vaginal lavage, crystal violet staining, and vaginal cytological evaluation for mouse estrous cycle staging identification.</a> J Vis Exp. (67), e4389 (2012).</li><li>JoVE Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lab+Animal+Research.+Fundamentals+of+Breeding+and+Weaning.">Lab Animal Research. Fundamentals of Breeding and Weaning.</a> JoVE. , (2017).</li><li>Prassas, I., Eissa, A., Poda, G., Diamandis, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unleashing+the+therapeutic+potential+of+human+kallikrein-related+serine+proteases.">Unleashing the therapeutic potential of human kallikrein-related serine proteases.</a> Nat Rev Drug Discov. 14 (3), 183-202 (2015).</li><li>Muytjens, C. M., Vasiliou, S. K., Oikonomopoulou, K., Prassas, I., Diamandis, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Putative+functions+of+tissue+kallikrein-related+peptidases+in+vaginal+fluid.">Putative functions of tissue kallikrein-related peptidases in vaginal fluid.</a> Nat Rev Urol. 13 (10), 596-607 (2016).</li></ol>

The authors have nothing to disclose.

Collecting semen from mouse uteri can provide advantages over semen analysis in vitro as the former can illustrate the physiological interactions between the semen and the secretions from the female reproductive tract. The techniques presented in this study allow researchers to determine semen quality as well as sperm viability and motility in ideal physiological conditions. Moreover, there are various downstream analyses that can be performed using the semen collected from the uteri, for example, the s...

The female reproductive tract is comprised of the upper and lower tracts. The upper reproductive tract includes Fallopian tubes, the uterus, and the endocervix. The lower reproductive tract includes the ectocervix and the vagina. In humans, the ejaculated semen is deposited at the anterior wall of the vagina, adjacent to the ectocervix1. In mice, however, the semen is swept into the uterus within minutes after mating2.
Semen contains seminal gel that is solidified within seconds of ejaculation, causing the sperm to be entrapped and immobilized3. Liquefaction releases the immobilized sperm by changing semen from a gel-like to a watery consistency. This process is essential for sperm motility and mammalian reproduction. Knowledge of semen liquefaction is mostly based on in vitro studies where semen becomes liquefied in a culture dish. In humans, the enzymatic activity of prostate specific antigen or PSA (also known as kallikrein-related peptidase 3 or KLK3) is the major contributor for liquefaction through hydrolysis of semenogelins (gel-forming proteins present in the semen)4,5,6. Degradation of semenogelins liberates the sperm from the seminal coagulum, increasing sperm mobility. Freed sperm swim toward the Fallopian tube to fertilize the egg. Liquefaction (or semen viscosity) tests are one of the standard initial screenings for semen analysis in fertility clinics to assess male fertility7.
A recently published article shows that analysis of seminal fluid collected from the female mouse reproductive tract post-mating can be used to illustrate a deficiency in liquefaction that can subsequently cause fertility defects8. Defective liquefaction such as semen hyperviscosity contributes to 11.8-32.3% of infertile cases in men9, suggesting that normal liquefaction is indispensable for mammalian reproduction. However, studies and treatment of liquefaction defects have focused exclusively on the male7. The possibility that the female reproductive tract plays a role in semen liquefaction has not been explored. Therefore, the methods for semen collection and measuring liquefaction time will provide researchers a novel diagnostic tool to determine whether liquefaction could be one of the causes of infertility in the model organism of interest.

<table border="0" cellpadding="0" cellspacing="0" width="571">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Toothpick</td>
			<td style="width:71px;">Diamond</td>
			<td style="width:119px;">750-Flat</td>
			<td style="width:167px;">Autoclaved sterile, use blunted toothpick. Do not use point-ended</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Dissecting scissors</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">08-940</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Spring scissors</td>
			<td style="width:71px;">Roboz</td>
			<td style="width:119px;">RS-5600</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Fine forceps</td>
			<td style="width:71px;">Roboz</td>
			<td style="width:119px;">RS-5045</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">25-&#956;L glass capillary tube</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">53432-761</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Leibovitz&#8217;s L-15 media</td>
			<td style="width:71px;">Life Technologies</td>
			<td style="width:119px;">11415064</td>
			<td style="width:167px;">Supplement with 1% FBS</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Fetal bovine serum (FBS)</td>
			<td style="width:71px;">Gemini Bio-Products</td>
			<td style="width:119px;">100-106</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">1.5-mL microcentrifuge tube</td>
			<td style="width:71px;">Axygen</td>
			<td style="width:119px;">MCT-150-C</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">2-mL microcentrifuge tube</td>
			<td style="width:71px;">Axygen</td>
			<td style="width:119px;">MCT-200-A</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:215px;">Digital dry block heater</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">13259-052</td>
			<td style="width:167px;">For warming the microcentrifuge tube prior to semen collection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Minicentrifuge</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">LSE 6765</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">35-mm culture dish</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">10861-656</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">15-mL polypropylene centrifuge tube</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">10025-686</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Waterbath</td>
			<td style="width:71px;">Precision</td>
			<td style="width:119px;">TSGP05</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Heated stage stereomicroscope</td>
			<td style="width:71px;">Leica Microsystems</td>
			<td style="width:119px;">MZ10F</td>
			<td style="width:167px;">To keep the tissue warm prior to semen collection</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Brightfield microscope</td>
			<td style="width:71px;">Leica Microsystems</td>
			<td style="width:119px;">DMi8</td>
			<td style="width:167px;">Optional. For video imaging of the sperm motility</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Camera</td>
			<td style="width:71px;">Leica Microsystems</td>
			<td style="width:119px;">DMC2900</td>
			<td style="width:167px;">Optional. For video imaging of the sperm motility</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Glass slides</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">12-552-3</td>
			<td style="width:167px;">Optional. For video imaging of the sperm motility</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Coverslip</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">48393-059</td>
			<td style="width:167px;">Optional. For video imaging of the sperm motility</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:215px;">Bleach</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Dilute in dH2O to 10% concentration</td>
		</tr>
	</tbody>
</table>

collection of post-mating semen from the female reproductive tract and measurement of semen liquefaction in mice

In mice, ejaculated semen is deposited in the uterus. After ejaculation, the semen changes consistency from gel-like to watery, a process called liquefaction. In this study, we show how to collect the post-ejaculated semen from the female reproductive tract in a mouse model. First, adult female mice in the estrus stage were housed in a male&#39;s cage overnight. The next morning, copulation was confirmed by the presence of copulatory plug at the vaginal opening. Female mice with copulatory plugs were euthanized, and each reproductive tract was collected as a whole (vagina, uterus, oviducts, ovaries), ensuring a closed system to contain the semen. The reproductive tract was placed in a 1.5 mL microcentrifuge tube, and the vagina was cut off to release the semen into the tube. To ensure maximum semen volume for analysis, toothless forceps were used to squeeze the uterine horns from ovarian end to vaginal end expelling remaining semen. The whole reproductive tract was then discarded. The semen-containing tube was briefly spun down. A 25 &#956;L capillary pipette was placed into the tube at a 180&#176; angle (parallel to the tube wall). The amount of time used to fill the capillary tube to the 25 &#956;L line was recorded. Semen from a proven male breeder usually takes approximately 60-180 s to fill a 25 &#956;L capillary tube. This semen collection technique can also be used in other downstream applications such as sperm imaging and motility analysis.

Semen was collected from CTRL and KO females approximately 8 h after mating with fertile CTRL males. Liquefaction (or semen viscosity) is quantified by the time taken to fill 25 &#956;L capillary tube with semen. Semen collected from CTRL uteri took 2.06 &#177;0.12 min (mean&#177;S.E.M, n = 5) to fill the capillary tube (Figure 1). However, the semen collected from KO uteri took longer than 60 min (60.0 &#177;0.0, mean&#177;S.E.M, n = 5) of experimental time. These data sug...

Watch this Scientific Journal Video about Collection of Post-mating Semen from the Female Reproductive Tract and Measurement of Semen Liquefaction in Mice at JoVE.com

Collection of Post-mating Semen from the Female Reproductive Tract and Measurement of Semen Liquefaction in Mice

Semen liquefaction is required for sperm to be liberated from the seminal gel. This study provides the procedures for collecting semen from the female reproductive tract post-coitus, and measuring semen liquefaction time.

In mice, ejaculated semen is deposited in the uterus. After ejaculation, the semen changes consistency from gel-like to watery, a process called ...

collection-post-mating-semen-from-female-reproductive-tract

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16.3K Views.  Washington State University. Semen liquefaction is required for sperm to be liberated from the seminal gel. This study provides the procedures for collecting semen from the female reproductive tract post-coitus, and measuring semen liquefaction time.

Video: Collection of Post-mating Semen from the Female Reproductive Tract and Measurement of Semen Liquefaction in Mice

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We quantitatively assessed follicle survival, measured follicle and oocyte diameters, and examined ultrastructure of the oocytes produced from the system. We observed decreased follicle survival, downregulation of expressions of proliferating cell nuclear antigen and growth differentiation factor 9, as indicators for the development of granulosa cells and oocytes, respectively, and oocyte ultrastructural abnormalities under the simulated microgravity condition. The simulated microgravity experimental setup needs to be optimized to provide a model for investigation of the mechanisms involved in the oocyte/follicle in vitro development.

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

A highly promising technique to generate tissue constructs without using matrix is to culture cells in a simulated microgravity condition. Using a rotary culture system, we examined ovarian follicle growth and oocyte maturation in terms of follicle survival, morphology, growth, and oocyte function under the simulated microgravity condition.

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Orthotopic Ovarian Transplantation Procedures to Investigate the Life- and Health-span Influence of Ovarian Senescence in Female Mice

Ovarian transplantation was first conducted at Utah State University in 1963. In more recent work, heterochronic transplantation of mammalian ovaries is being used to investigate the health-protective effects of young ovaries in young females. The current procedures employ an orthotopic transplantation method, where allogenic ovaries are transplanted back to their original position in the ovarian bursa. This is in contrast to the more commonly used heterotopic transplantation of ovaries/ovarian tissue subcutaneously or under the kidney capsule. All three locations provide efficient revascularization of the transplanted tissues. However, orthotopic transplantation provides the ovary with the most natural signaling environment and is the only procedure that provides the opportunity for the animal to reproduce naturally post-operatively. One must take care to remove all endogenous ovarian tissue during the ovariectomy procedure. If any endogenous tissue remains or if only one ovary is removed, the transplanted tissue will remain dormant until the existing tissue becomes senescent. While revascularization of the transplanted ovaries occurs very quickly, the transplant recipient can take a considerable amount of time to adapt to a new hypothalamic/pituitary/gonadal/adrenal (HPG/A) axis signaling regime associated with the transplanted tissue. This normally takes about 100 days in the mouse. Therefore, transplantation experiments should be designed to accommodate this adaptation period. Typical results with ovarian transplantation will include changes in the health of the recipient that reflect the age of the transplanted ovary, rather than the chronological age of the recipient.

Described here are orthotopic ovarian transplantation protocols for modifying ovarian influence on the physiology of the recipient mouse. This surgical procedure is used to expose female mice to various types of ovarian influence and may include heterochronic transplantations and transplantation of ovaries that have undergone various ex vivo treatments.

Ovarian transplantation was first conducted at Utah State University in 1963. In more recent work, heterochronic transplantation of mammalian ovaries is ...

Combinational Treatment of Trichostatin A and Vitamin C Improves the Efficiency of Cloning Mice by Somatic Cell Nuclear Transfer

Somatic cell nuclear transfer (SCNT) provides a unique opportunity to directly produce a cloned animal from a donor cell, and it requires the use of skillful techniques. Additionally, the efficiencies of cloning have remained low since the successful production of cloned animals, especially mice. There have been many attempts to improve the cloning efficiency, and trichostatin A (TSA), a histone deacetylase inhibitor, has been widely used to enhance the efficiency of cloning. Here, we report a dramatically improved cloning method in mice. This somatic cell nuclear transfer method involves usage of Hemagglutinating virus of Japan Envelope (HVJ-E), which enables easy manipulation. Moreover, the treatment using two small molecules, TSA and vitamin C (VC), with deionized bovine serum albumin (dBSA), is highly effective for embryonic development. This approach requires neither additional injection nor genetic manipulation, and thus presents a simple, suitable method for practical use. This method could become a technically feasible approach for researchers to produce genetically modified animals from cultured cells. Furthermore, it might be a useful way for the rescue of endangered animals via cloning.

We describe a dramatically improved method for mouse cloning using trichostatin A, vitamin C, and deionized bovine serum albumin. We show a simplified, reproducible protocol that supports efficient development of cloned embryos. Hence, this method could become a standardized procedure for mouse cloning.

Somatic cell nuclear transfer (SCNT) provides a unique opportunity to directly produce a cloned animal from a donor cell, and it requires the use of ...

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

The differentiation of human endometrial stromal cells (HESC) from fibroblast-like appearance into secretory decidua is a transformation required for embryo implantation into the uterine lining of the maternal womb. Improper decidualization has been established as a root cause for implantation failure and subsequent early embryo miscarriage. Therefore, understanding the molecular mechanisms underlying decidualization is advantageous to improving the rate of successful births. In vivo based studies of artificial decidualization are often limiting due to ethical dilemmas associated with human research, as well as translational complications within animal models. As a result, in vitro assays through primary cell culture are often utilized to explore the modulation of decidualization via hormones. This study provides a detailed protocol for the isolation of HESC and subsequent artificial decidualization via the supplementation of hormones to the culturing medium. Further, this study provides a well-designed method to knockdown any gene of interest by utilizing lipid-based siRNA transfections. This protocol permits the optimization of culture purity as well as product yield, thereby maximizing the ability to utilize this model as a reliable method to understand the molecular mechanisms underlying decidualization, and the subsequent quantification of secreted agents by decidualized endometrial stromal cells.

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

This study presents a validated and optimized procedure for the isolation and culture of human endometrial stromal cells to conduct in vitro decidualization assay. Further, this study provides a detailed method to efficiently knockdown a specific gene using siRNAs in human endometrial stromal cells.

The differentiation of human endometrial stromal cells (HESC) from fibroblast-like appearance into secretory decidua is a transformation required for ...

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by Sertoli cell junctions at the blood-testis barrier (BTB), which is the tightest tissue barrier in the mammalian body and segregates the seminiferous epithelium into two compartments, a basal and an adluminal. The BTB creates a unique microenvironment for germ cells in meiosis I/II and for the development of postmeiotic spermatids into spermatozoa via spermiogenesis. Here, we describe a reliable assay to monitor BTB integrity of mouse testis in vivo. An intact BTB blocks the diffusion of FITC-conjugated inulin from the basal to the apical compartment of the seminiferous tubules. This technique is suitable for studying gene candidates, viruses, or environmental toxicants that may affect BTB function or integrity, with an easy procedure and a minimal requirement of surgical skills compared to alternative methods.

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Here, we present a protocol to assess the blood-testis barrier integrity by injecting inulin-FITC into testes. This is an efficient in vivo method to study blood-testis barrier integrity that can be compromised by genetic and environmental elements.

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by ...

In Vitro Assays to Evaluate the Migration, Invasion, and Proliferation of Immortalized Human First-trimester Trophoblast Cell Lines

Cell movement is a critical property of trophoblasts during placental development and early pregnancy. The significance of proper trophoblast migration and invasion is demonstrated by pregnancy disorders such as pre-eclampsia and intrauterine growth restriction, which are associated with inadequate trophoblast invasion of the maternal vasculature. Unfortunately, our understanding of the mechanisms by which the placenta develops from migrating trophoblasts is limited. In vitro analysis of cell migration via the scratch assay is a useful tool in identifying factors that regulate trophoblast migratory capacity. However, this assay alone does not define the cellular changes that can result in altered cell migration. This protocol describes three different in vitro assays that are used collectively to evaluate trophoblast cell movement: the scratch assay, the invasion assay, and the proliferation assay. The protocols described here may also be modified for use in other cell lines to quantify cell movement in response to stimuli. These methods allow investigators to identify individual factors that contribute to the cell movement and provide a thorough examination of potential mechanisms underlying apparent changes in cell migration.

Here, we present a highly accessible protocol for evaluating the cell movement in human trophoblast cells using three in vitro assays: the scratch assay, the transwell invasion assay, and the cell proliferation assay.

Cell movement is a critical property of trophoblasts during placental development and early pregnancy. The significance of proper trophoblast migration ...

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 ...

In Vitro Culture Strategy for Oocytes from Early Antral Follicle in Cattle

The limited reserve of mature, fertilizable oocytes represents a major barrier for the success of assisted reproduction in mammals. Considering that during the reproductive life span only about 1% of the oocytes in an ovary mature and ovulate, several techniques have been developed to increase the exploitation of the ovarian reserve to the growing population of non-ovulatory follicles. Such technologies have allowed interventions of fertility preservation, selection programs in livestock, and conservation of endangered species. However, the vast potential of the ovarian reserve is still largely unexploited. In cows, for instance, some attempts have been made to support in vitro culture of oocytes at specific developmental stages, but efficient and reliable protocols have not yet been developed. Here we describe a culture system that reproduce the physiological conditions of the corresponding follicular stage, defined to develop in vitro growing oocytes collected from bovine early antral follicles to the fully-grown stage, corresponding to the medium antral follicle in vivo. A combination of hormones and a phosphodiesterase 3 inhibitor was used to prevent untimely meiotic resumption and to guide oocyte&#39;s differentiation.

We describe the procedures for isolation of growing oocytes from ovarian follicles at early stages of development, as well as the setup of an in vitro culture system which can support the growth and differentiation up to the fully-grown stage. &#160;

The limited reserve of mature, fertilizable oocytes represents a major barrier for the success of assisted reproduction in mammals. Considering that ...

Transuterine Fetal Tracheal Occlusion Model in Mice

Fetal tracheal occlusion (TO), an established treatment modality, promotes fetal lung growth and survival in severe congenital diaphragmatic hernia (CDH). Following TO, retention of the secreted epithelial fluid increases luminal pressure and induces lung growth. Various animal models have been defined to understand the pathophysiology of CDH and TO. All have their own advantages and disadvantages such as the difficulty of the technique, the size of the animal, cost, high mortality rates, and the availability of genetic tools. Herein, a novel transuterine model of murine fetal TO is described. Pregnant mice were anesthetized, and the uterus exposed via a midline laparotomy. The trachea of selected fetuses were ligated with a single transuterine suture placed behind the trachea, one carotid artery, and one jugular vein. The dam was closed and allowed to recover. Fetuses were collected just before parturition. Lung to body weight ratio in TO fetuses was higher than that in control fetuses. This model provides researchers with a new tool to study the impact of both TO and increased luminal pressure on lung development.

Various animal models of congenital diaphragmatic hernia and fetal tracheal occlusion present advantages and disadvantages regarding ethical issues, cost, surgical difficulty, size, survival rates, and availability of genetic tools. This model provides a new tool to study the impact of both tracheal occlusion and increased luminal pressure on lung development.

Fetal tracheal occlusion (TO), an established treatment modality, promotes fetal lung growth and survival in severe congenital diaphragmatic hernia (CDH). ...