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><tbody><tr><td>Toothpick</td><td>Diamond</td><td>750-Flat</td><td>Autoclaved sterile, use blunted toothpick. Do not use point-ended</td></tr><tr><td>Dissecting scissors</td><td>Fisher</td><td>08-940</td><td></td></tr><tr><td>Spring scissors</td><td>Roboz</td><td>RS-5600</td><td></td></tr><tr><td>Fine forceps</td><td>Roboz</td><td>RS-5045</td><td></td></tr><tr><td>25-&amp;mu;L glass capillary tube</td><td>VWR</td><td>53432-761</td><td></td></tr><tr><td>Leibovitz&amp;rsquo;s L-15 media</td><td>Life Technologies</td><td>11415064</td><td>Supplement with 1% FBS</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Gemini Bio-Products</td><td>100-106</td><td></td></tr><tr><td>1.5-mL microcentrifuge tube</td><td>Axygen</td><td>MCT-150-C</td><td></td></tr><tr><td>2-mL microcentrifuge tube</td><td>Axygen</td><td>MCT-200-A</td><td></td></tr><tr><td>Digital dry block heater</td><td>VWR</td><td>13259-052</td><td>For warming the microcentrifuge tube prior to semen collection</td></tr><tr><td>Minicentrifuge</td><td>Corning</td><td>LSE 6765</td><td></td></tr><tr><td>35-mm culture dish</td><td>VWR</td><td>10861-656</td><td></td></tr><tr><td>15-mL polypropylene centrifuge tube</td><td>VWR</td><td>10025-686</td><td></td></tr><tr><td>Waterbath</td><td>Precision</td><td>TSGP05</td><td></td></tr><tr><td>Heated stage stereomicroscope</td><td>Leica Microsystems</td><td>MZ10F</td><td>To keep the tissue warm prior to semen collection</td></tr><tr><td>Brightfield microscope</td><td>Leica Microsystems</td><td>DMi8</td><td>Optional. For video imaging of the sperm motility</td></tr><tr><td>Camera</td><td>Leica Microsystems</td><td>DMC2900</td><td>Optional. For video imaging of the sperm motility</td></tr><tr><td>Glass slides</td><td>Fisher</td><td>12-552-3</td><td>Optional. For video imaging of the sperm motility</td></tr><tr><td>Coverslip</td><td>VWR</td><td>48393-059</td><td>Optional. For video imaging of the sperm motility</td></tr><tr><td>Bleach</td><td></td><td></td><td>Dilute in dH&lt;sub&gt;2&lt;/sub&gt;O 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

Research

JoVE Journal

Developmental Biology

16.4K Views.  Washington State University. Sperma vloeibaar is vereist voor sperma worden bevrijd van de rudimentaire gel. Deze studie bevat de procedures voor het verzamelen van sperma van de vrouwelijke voortplantingssysteem na coïtus en sperma vloeibaar tijdmeting.

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

Harvesting Sperm and Artificial Insemination of Mice

Rodents of the genus Peromyscus (deer mice) are the most prevalent native North American mammals. Peromyscus species are used in a wide range of research including toxicology, epidemiology, ecology, behavioral, and genetic studies. Here they provide a useful model for demonstrations of artificial insemination. 

Methods similar to those displayed here have previously been used in several deer mouse studies, yet no detailed protocol has been published. Here we demonstrate the basic method of artificial insemination. This method entails extracting the testes from the rodent, then isolating the sperm from the epididymis and vas deferens. The mature sperm, now in a milk mixture, are placed in the female&#x2019;s reproductive tract at the time of ovulation. Fertilization is counted as day 0 for timing of embryo development. Embryos can then be retrieved at the desired time-point and manipulated.

Artificial insemination can be used in a variety of rodent species where exact embryo timing is crucial or hard to obtain. This technique is vital for species or strains (including most Peromyscus) which may not mate immediately and/or where mating is hard to assess. In addition, artificial insemination provides exact timing for embryo development either in mapping developmental progress and/or transgenic work. Reduced numbers of animals can be used since fertilization is guaranteed. This method has been vital to furthering the Peromyscus system, and will hopefully benefit others as well.

This protocol demonstrates methods for extracting sperm from the testes of males and then inseminating female mice. This procedure is useful when precise time is needed in developmental studies as well as transgenic work.

Rodents of the genus Peromyscus (deer mice) are the most prevalent native North American mammals.  Peromyscus species are used in a wide range of research ...

Biology

Assessment of Sexual Behavior of Male Mice

Sexual behavior is highly species-specific. Although rodents have slightly different sexual behaviors, mice and rats have a similar sexual behavioral pattern. The purpose of this article is to describe the hormone-induced estrus ovariectomized female model and the experimental procedure for the assessment of sexual behavior of male mice. The most important sexual behavioral elements are demonstrated in the video and illustrations. The critical steps, advantages, and limitations of the sexual behavior test are explained as well. Finally, the behavior parameters are presented, and mounting, intromission, and ejaculation processes in mating are distinguished. Behavioral parameters are assessed in terms of the occurred duration and counts during the test period.

This article describes how to perform sexual behavior tests in male mice.

Sexual behavior is highly species-specific. Although rodents have slightly different sexual behaviors, mice and rats have a similar sexual behavioral ...

Behavior

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Immunology and Infection

Biaxial Basal Tone and Passive Testing of the Murine Reproductive System Using a Pressure Myograph

The female reproductive organs, specifically the vagina and cervix, are composed of various cellular components and a unique extracellular matrix (ECM). Smooth muscle cells exhibit a contractile function within the vaginal and cervical walls. Depending on the biochemical environment and the mechanical distension of the organ walls, the smooth muscle cells alter the contractile conditions. The contribution of the smooth muscle cells under baseline physiological conditions is classified as a basal tone. More specifically, a basal tone is the baseline partial constriction of smooth muscle cells in the absence of hormonal and neural stimulation. Furthermore, the ECM provides structural support for the organ walls and functions as a reservoir for biochemical cues. These biochemical cues are vital to various organ functions, such as inciting growth and maintaining homeostasis. The ECM of each organ is composed primarily of collagen fibers (mostly collagen types I, III, and V), elastic fibers, and glycosaminoglycans/proteoglycans. The composition and organization of the ECM dictate the mechanical properties of each organ. A change in ECM composition may lead to the development of reproductive pathologies, such as pelvic organ prolapse or premature cervical remodeling. Furthermore, changes in ECM microstructure and stiffness may alter smooth muscle cell activity and phenotype, thus resulting in the loss of the contractile force.
In this work, the reported protocols are used to assess the basal tone and passive mechanical properties of the nonpregnant murine vagina and cervix at 4-6 months of age in estrus. The organs were mounted in a commercially available pressure myograph and both pressure-diameter and force-length tests were performed. Sample data and data analysis techniques for the mechanical characterization of the reproductive organs are included. Such information may be useful for constructing mathematical models and rationally designing therapeutic interventions for women&#8217;s health pathologies.

This protocol utilized a commercially available pressure myograph system to perform pressure myograph testing on the murine vagina and cervix. Utilizing media with and without calcium, the contributions of the smooth muscle cells (SMC) basal tone and passive extracellular matrix (ECM) were isolated for the organs under estimated physiological conditions.

The female reproductive organs, specifically the vagina and cervix, are composed of various cellular components and a unique extracellular matrix (ECM). ...

Bioengineering

Performing Vaginal Lavage, Crystal Violet Staining, and Vaginal Cytological Evaluation for Mouse Estrous Cycle Staging Identification

A rapid means of assessing reproductive status in rodents is useful not only in the study of reproductive dysfunction but is also required for the production of new mouse models of disease and investigations into the hormonal regulation of tissue degeneration (or regeneration) following pathological challenge. The murine reproductive (or estrous) cycle is divided into 4 stages: proestrus, estrus, metestrus, and diestrus. Defined fluctuations in circulating levels of the ovarian steroids 17-&beta;-estradiol and progesterone, the gonadotropins luteinizing and follicle stimulating hormones, and the luteotropic hormone prolactin signal transition through these reproductive stages. Changes in cell typology within the murine vaginal canal reflect these underlying endocrine events. Daily assessment of the relative ratio of nucleated epithelial cells, cornified squamous epithelial cells, and leukocytes present in vaginal smears can be used to identify murine estrous stages. The degree of invasiveness, however, employed in collecting these samples can alter reproductive status and elicit an inflammatory response that can confound cytological assessment of smears. Here, we describe a simple, non-invasive protocol that can be used to determine the stage of the estrous cycle of a female mouse without altering her reproductive cycle. We detail how to differentiate between the four stages of the estrous cycle by collection and analysis of predominant cell typology in vaginal smears and we show how these changes can be interpreted with respect to endocrine status.

Here, we describe how to identify the stage of the murine reproductive (proestrus, estrus, metestrus, or diestrus) by simple, non-invasive collection and cytological assessment of vaginal smear samples. We further describe how vaginal cytology reflects circulating hormonal levels underlying transition through the murine reproductive cycle.

A rapid means of assessing reproductive status in rodents is useful not only in the study of reproductive dysfunction but is also required for the ...

Determination of Reproductive Competence by Confirming Pubertal Onset and Performing a Fertility Assay in Mice and Rats

Assessment of reproductive competence is critical for understanding the impact of a treatment or genetic manipulation on the reproductive axis, also termed the hypothalamic-pituitary-gonadal axis. The reproductive axis is a key integrator of environmental and internal input adapting fertility to favorable conditions for reproduction. Prior to embarking upon a fertility study in mice and rats, sexual maturity is evaluated to exclude the possibility that the observed reproductive phenotypes are caused by delayed or absent pubertal onset. This protocol describes a non-invasive approach to assess pubertal onset in males through the determination of preputial separation, and in females through vaginal opening and first estrus. After the confirmation of the completion of puberty and the achievement of sexual maturity, a fertility study can be initiated. The procedure describes the optimal breeding conditions for mice and rats, how to set up a fertility study, and what parameters to evaluate and determine if the treatment or gene deletion has an impact on fertility.

Many treatments and genetic mutations impact the timing of sexual maturity and fertility. This protocol describes a non-invasive method to evaluate pubertal onset in mice and rats prior to setting up a fertility study in sexually mature animals.

Assessment of reproductive competence is critical for understanding the impact of a treatment or genetic manipulation on the reproductive axis, also ...

Ex Vivo Method for Assessing the Mouse Reproductive Tract Spontaneous Motility and a MATLAB-based Uterus Motion Tracking Algorithm for Data Analysis

Dysmenorrhea, or painful cramping, is the most common symptom associated with menses in females and its severity can hinder women&#39;s everyday lives. Here, we present an easy and inexpensive method that would be instrumental for testing new drugs decreasing uterine contractility. This method utilizes the unique ability of the entire mouse reproductive tract to exhibit spontaneous motility when maintained ex vivo in a Petri dish containing oxygenated Krebs buffer. This spontaneous motility resembles the wave-like myometrial activity of the human uterus, referred to as endometrial waves. To demonstrate the effectiveness of the method, we employed a well-known uterine relaxant drug, epinephrine. We demonstrate that the spontaneous motility of the entire mouse reproductive tract can be quickly and reversibly inhibited by 1 &#181;M epinephrine in this Petri dish model. Documenting the changes of uterine motility can be easily done using an ordinary smart phone or a sophisticated digital camera. We developed a MATLAB-based algorithm allowing motion tracking to quantify spontaneous uterine motility changes by measuring the rate of uterine horn movements. A major advantage of this ex vivo approach is that the reproductive tract remains intact throughout the entire experiment, preserving all intrinsic intrauterine cellular interactions. The major limitation of this approach is that up to 10-20% of uteri may exhibit no spontaneous motility. Thus far, this is the first quantitative ex vivo method for assessing spontaneous uterine motility in a Petri dish model.

Uterine contractions are important for the well-being of females. However, pathologically increased contractility may result in dysmenorrhea, especially in younger females. Here, we describe a simple ex vivo preparation allowing quick assessment of the efficacy of smooth muscle relaxants that may be used for treating dysmenorrhea.

Dysmenorrhea, or painful cramping, is the most common symptom associated with menses in females and its severity can hinder women&#39;s everyday lives. ...

Development of a Novel Endometrioma-Simulating Mouse Model

Establishment of an Experimental Mouse Model of Endometrioma to Study its Related Infertility

Endometrioma (OMA), a subtype of endometriosis characterized by the formation of endometriotic cysts in the ovaries, affects 17-44% of individuals diagnosed with endometriosis. Women with OMA often experience compromised fertility, yet the exact mechanisms underlying OMA-associated infertility remain unclear. Notably, existing animal models simulate superficial peritoneal endometriosis (SUP) and deep infiltrating endometriosis (DIE), leaving a notable gap in research focused on OMA. In response to the gap of knowledge, this paper introduces a pioneering OMA-simulating mouse model and provides a comprehensive description of the techniques and procedures employed in the model. With a high success rate of 83% and ovarian lesion specificity, this model holds significant promise for advancing our understanding of OMA, particularly in the context of infertility. It offers a valuable platform for conducting targeted research into OMA-associated fertility challenges, potentially paving the way for improved diagnostic and therapeutic strategies in the field of reproductive medicine.

This study introduces a novel mouse model specifically designed to simulate endometrioma, a clinically significant subtype of endometriosis. By utilizing C57BL/6J mice, the model aims to elucidate the pathophysiological mechanisms underlying endometrioma-related infertility, offering a refined tool to bridge the current knowledge gap in reproductive medicine.

Endometrioma (OMA), a subtype of endometriosis characterized by the formation of endometriotic cysts in the ovaries, affects 17-44% of individuals ...

Medicine

Cervical Manipulation Induced Pseudopregnancy in Mice for Assisted Reproduction

Inducing Pseudopregnancy in Female Mice Without the Need for Vasectomized Males Prior to Non-Surgical Embryo Transfer or Artificial Insemination

For successfully maintaining pregnancy with embryo transfer or artificial insemination,&#160;female recipient mice must be induced into a pseudopregnant state. Female mice are traditionally paired overnight with vasectomized males, and the following morning, the presence of a copulation plug is assessed. To increase the efficiency of producing pseudopregnant females, a cervical manipulation technique has been standardized to be used in combination with non-surgical embryo transfer or artificial insemination techniques in mice. The blunt end of a small plastic rod is inserted vaginally to contact the cervix and is vibrated for 30 s by contact with a trimmer. The procedure is quick and does not require anesthesia or analgesia. This technique increases the reliability and predictability of producing pseudopregnant females and entirely eliminates the requirement for vasectomized males. For CD1 mice, the efficiency of pseudopregnancy induction using cervical manipulation was 83% for females in estrus (N = 76) but only 38% of females in estrus were plugged by vasectomized males (N = 24). Artificial insemination in CD1 mice was performed by estrus synchronization with hormones, cervical manipulation, and the uterine transfer of sperm. Artificial insemination recipients receiving cervical manipulation (N = 76) had a pregnancy rate of 72% and an average litter size of 8.3 pups. This method can also be used to produce pseudopregnant females for non-surgical embryo transfer. Therefore, inducing pseudopregnancy by cervical manipulation is a convenient and efficient alternative to mating with a vasectomized male when performing non-surgical assisted reproduction techniques. Using cervical manipulation provides 3Rs (replacement, reduction, and refinement) benefits for assisted reproduction techniques by reducing the number of animals required and eliminating the necessity for surgically altered males.

Author Spotlight: Inducing Pseudopregnancy in Female Mice Without the Need for Vasectomized Males Prior to Non-Surgical Embryo Transfer or Artificial Insemination  

The cervical manipulation method presented can induce pseudopregnancy in mice without the need for breeding females with vasectomized males. The induction of pseudopregnancy is required for the success of non-surgical embryo transfer and non-surgical artificial insemination, both of which are also presented.

For successfully maintaining pregnancy with embryo transfer or artificial insemination,&#160;female recipient mice must be induced into a pseudopregnant ...

Optimized Thin Endometrium Mouse Model with Low Mortality and Minimal Adhesions

Establishing a Mouse Model of Thin Endometrium

Thin endometrium (TE) has been widely recognized as a critical cause of infertility. However, the pathogenesis of TE remains unclear, and satisfactory treatment options are still urgently needed. Several animal models of TE have been developed, but the mouse model involving abdominal surgery and injection of 95% ethanol presents a formidable challenge due to the high mortality rate and risk of intrauterine adhesions if not performed correctly. Here, we describe a detailed protocol that produces reliable and reproducible TE with a very low mortality rate and minimal intrauterine adhesions by injecting 95% ethanol into the mouse uterus with varying infusion times. The results showed that all of the mice successfully developed TE with infusion times ranging from 1-3 min, characterized by a typical reduction in endometrial thickness and the number of glands, as well as excessive endometrial fibrosis. These findings suggest that this mouse model is suitable for studying thin endometrium and can serve as a platform for developing future TE treatments.

This article describes a detailed protocol for producing a reliable and reproducible thin endometrium with a very low mortality rate and minimal intrauterine adhesions by injecting 95% ethanol into the mouse uterus within 1-3 min.

Thin endometrium (TE) has been widely recognized as a critical cause of infertility. However, the pathogenesis of TE remains unclear, and satisfactory ...

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

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