Name: methods for studying uterine contributions to pregnancy establishment in an ovariectomized mouse model
Uploaded: 2023-04-07T12:50:00
Description: Watch this Scientific Journal Video about Methods for Studying Uterine Contributions to Pregnancy Establishment in an Ovariectomized Mouse Model at JoVE.com

This work was made possible through the Victorian State Government Operational Infrastructure Support and Australian Government National Health and Medical Research Council (NHMRC) IRIISS. This work was supported by the Monash University Faculty of Medicine, Nursing and Health Science Platform Access Grant to A.L.W. (Winship-PAG18-0343) to access the Monash Reproductive Services Platform. A.L.W. is supported by DECRA funding DE21010037 from the Australian Research Council (ARC). J.N.H. and L.R.A. are supported by an Australian Government Research Training Program Scholarship. L.R.A. is supported by a Monash Graduate Excellence Scholarship. K.J.H. is supported by an ARC Future Fellowship FT190100265.

All animals were housed in temperature-controlled, high-barrier facilities (Monash University Animal Research Laboratory) with free food and water access and a 12 h light-dark cycle. All the procedures were performed in accordance with approval from the Monash Animal Research Platform Ethics committee (#21908, 17971) and performed in accordance with the National Health and Medical Research Council Code of Practice for the care and use of animals.
1. Surgical preparation
<ol>...

<ol>
	<li>Szamatowicz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assisted+reproductive+technology+in+reproductive+medicine+-+Possibilities+and+limitations.">Assisted reproductive technology in reproductive medicine - Possibilities and limitations.</a> Ginekologia Polska. 87 (12), 820-823 (2016).</li><li>Evans, 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=Fertile+ground:+Human+endometrial+programming+and+lessons+in+health+and+disease.">Fertile ground: Human endometrial programming and lessons in health and disease.</a> Nature Reviews. Endocrinology. 12 (11), 654-667 (2016).</li><li>Norwitz, E. R., Schust, D. J., Fisher, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implantation+and+the+survival+of+early+pregnancy.">Implantation and the survival of early pregnancy.</a> The New England Journal of Medicine. 345 (19), 1400-1408 (2001).</li><li>Zinaman, M. J., Clegg, E. D., Brown, C. C., O'Connor, J., Selevan, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimates+of+human+fertility+and+pregnancy+loss.">Estimates of human fertility and pregnancy loss.</a> Fertility &amp; Sterility. 65 (3), 503-509 (1996).</li><li>Kupka, M. S., 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=Assisted+reproductive+technology+in+Europe,+2010:+Results+generated+from+European+registers+by+ESHRE&#8224;.">Assisted reproductive technology in Europe, 2010: Results generated from European registers by ESHRE&#8224;.</a> Human Reproduction. 29 (10), 2099-2113 (2014).</li><li>Gleicher, N., Kushnir, V. A., Barad, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Worldwide+decline+of+IVF+birth+rates+and+its+probable+causes.">Worldwide decline of IVF birth rates and its probable causes.</a> Human Reproduction Open. 2019 (3), (2019).</li><li>Diaz-Gimeno, P., 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=A+genomic+diagnostic+tool+for+human+endometrial+receptivity+based+on+the+transcriptomic+signature.">A genomic diagnostic tool for human endometrial receptivity based on the transcriptomic signature.</a> Fertility &amp; Sterility. 95 (1), 50-60 (2011).</li><li>Amin, 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=Personalized+embryo+transfer+outcomes+in+recurrent+implantation+failure+patients+following+endometrial+receptivity+array+with+pre-implantation+genetic+testing.">Personalized embryo transfer outcomes in recurrent implantation failure patients following endometrial receptivity array with pre-implantation genetic testing.</a> Cureus. 14 (6), e26248 (2022).</li><li>Patel, J. A., Patel, A. J., Banker, J. M., Shah, S. I., Banker, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Personalized+embryo+transfer+helps+in+improving+in+vitro+fertilization/ICSI+outcomes+in+patients+with+recurrent+implantation+failure.">Personalized embryo transfer helps in improving in vitro fertilization/ICSI outcomes in patients with recurrent implantation failure.</a> Journal of Human Reproductive Sciences. 12 (1), 59-66 (2019).</li><li>Khan, K. N., 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=Biological+differences+between+functionalis+and+basalis+endometria+in+women+with+and+without+adenomyosis.">Biological differences between functionalis and basalis endometria in women with and without adenomyosis.</a> European Journal of Obstetrics, Gynecology, and Reproductive Biology. 203, 49-55 (2016).</li><li>Richards, J. S., Ren, Y. A., Candelaria, N., Adams, J. E., Rajkovic, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+follicular+theca+cell+recruitment,+differentiation,+and+impact+on+fertility:+2017+update.">Ovarian follicular theca cell recruitment, differentiation, and impact on fertility: 2017 update.</a> Endocrine Reviews. 39 (1), 1-20 (2018).</li><li>Corciulo, 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=Pulsed+administration+for+physiological+estrogen+replacement+in+mice.">Pulsed administration for physiological estrogen replacement in mice.</a> F1000Research. 10, 809 (2021).</li><li>Greaves, 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=A+novel+mouse+model+of+endometriosis+mimics+human+phenotype+and+reveals+insights+into+the+inflammatory+contribution+of+shed+endometrium.">A novel mouse model of endometriosis mimics human phenotype and reveals insights into the inflammatory contribution of shed endometrium.</a> The American Journal of Pathology. 184 (7), 1930-1939 (2014).</li><li>Griffiths, M. J., Alesi, L. R., Winship, A. L., Hutt, K. J. <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+an+embryo+transfer+model+to+study+uterine+contributions+to+pregnancy+in+vivo+in+mice.">Development of an embryo transfer model to study uterine contributions to pregnancy in vivo in mice.</a> Reproduction &amp; Fertility. 3 (1), 10-18 (2022).</li><li>Cousins, F. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+from+a+mouse+model+that+epithelial+cell+migration+and+mesenchymal-epithelial+transition+contribute+to+rapid+restoration+of+uterine+tissue+integrity+during+menstruation.">Evidence from a mouse model that epithelial cell migration and mesenchymal-epithelial transition contribute to rapid restoration of uterine tissue integrity during menstruation.</a> PLoS One. 9 (1), e86378 (2014).</li><li>Cousins, F. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Androgens+regulate+scarless+repair+of+the+endometrial+&amp;#34;wound&amp;#34;+in+a+mouse+model+of+menstruation.">Androgens regulate scarless repair of the endometrial &amp;#34;wound&amp;#34; in a mouse model of menstruation.</a> FASEB Journal. 30 (8), 2802-2811 (2016).</li><li>Fullerton, P. T., Monsivais, D., Kommagani, R., Matzuk, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Follistatin+is+critical+for+mouse+uterine+receptivity+and+decidualization.">Follistatin is critical for mouse uterine receptivity and decidualization.</a> Proceedings of the National Academy of Sciences of the United States of America. 114 (24), E4772-E4781 (2017).</li><li>Rowland, R. R., Reyes, E., Chukwuocha, R., Tokuda, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corticosteroid+and+immune+responses+of+mice+following+mini-osmotic+pump+implantation.">Corticosteroid and immune responses of mice following mini-osmotic pump implantation.</a> Immunopharmacology. 20 (3), 187-190 (1990).</li><li>Barton, B. 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=Roles+of+steroid+hormones+in+oviductal+function.">Roles of steroid hormones in oviductal function.</a> Reproduction. 159 (3), R125-R137 (2020).</li><li>Lee, J. 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=Autophagy+regulates+embryonic+survival+during+delayed+implantation.">Autophagy regulates embryonic survival during delayed implantation.</a> Endocrinology. 152 (5), 2067-2075 (2011).</li><li>Hamatani, T., 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=Global+gene+expression+analysis+identifies+molecular+pathways+distinguishing+blastocyst+dormancy+and+activation.">Global gene expression analysis identifies molecular pathways distinguishing blastocyst dormancy and activation.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (28), 10326-10331 (2004).</li><li>Cui, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcervical+embryo+transfer+in+mice.">Transcervical embryo transfer in mice.</a> Journal of the American Association for Laboratory Animal Science. 53 (3), 228-231 (2014).</li></ol>

This article provides step-by-step instructions on how to perform OVX and provide exogenous hormones for studies focused on understanding the uterine contributions to pregnancy and fertility. Two detailed protocols are provided on two experimental applications of these methods, including performing embryo transfer and inducing decidualization artificially.
Whilst performing OVX can be challenging initially - especially for researchers new to rodent models - it is a relatively simple procedure ...

With the rising use of assisted reproductive technologies in recent decades, many barriers to conception have been overcome, allowing many couples to start families despite fertility problems1. Oocyte or sperm deficits can often be bypassed using in vitro fertilization or intracytoplasmic sperm injection; however, issues related to the uterus and endometrial receptivity remain an elusive &#34;black box&#34; of reproductive potential2.
Pregnancy is established when a high-quality embryo successfully interacts with a receptive endometrium (uterine lining). The chances of successful pregnancy in any given menstrual cycle are low, at around 30%3,4. Of those that are successful, only 50%-60% advance past 20 weeks of gestation, with implantation failure being responsible for 75% of pregnancies that do not reach 20 weeks3. Despite these figures dating back to the late 1990s, the field is yet to overcome the limitations caused by an inadequately receptive endometrium. This has resulted in stagnating - and sometimes declining - IVF success rates in recent years5,6.
Women with unexplained infertility often have a displaced window of receptivity or are unable to achieve receptivity for unknown reasons. Recently, the endometrial receptivity array was developed, which assesses the expression of hundreds of genes with the purpose of tailoring the timing of embryo transfer to an individual&#39;s window of receptivity7,8,9. However, the field still lacks an understanding of the pathogenesis of pregnancy complications that manifest after the implantation process is complete.
The female reproductive system is highly dynamic and under tight hormonal control. The hypothalamic-pituitary-gonadal (HPG) axis controls the release of luteinizing hormone and follicle-stimulating hormone, which regulate aspects of the ovarian cycle, including follicle maturation and estrogen and progesterone activity. In turn, the uterine menstrual cycle is regulated by estrogens and progesterone10,11. Thus, studying uterine biological mechanisms is complicated by ovarian influence. For example, when studying how cancer therapies may impact the uterus, it can be difficult to distinguish if any uterine phenotype observed (such as pregnancy loss or menstrual acyclicity) is the result of a direct insult to the uterus or a consequential effect from damage to the ovaries.
To comprehensively understand fertility, the uterine contributions to pregnancy must be characterized. Importantly, this understanding must extend beyond uterine function under ovarian control. This cannot be studied in humans; therefore, animal models are often employed. As such, ovariectomy (OVX) is commonly used to enable researchers to regulate rodent estrous cycles (analogous to the menstrual cycle) by supplying hormones exogenously. Additionally, OVX allows uterine responses to be studied independently of ovarian influence12. However, if hormones are not immediately supplied post-OVX, a menopause phenotype will eventuate, which needs to be carefully considered by the researchers.
OVX is frequently utilized in rodent models13,14,15,16,17 and is relatively easy to perform after adequate training. Methods vary depending on whether the ovary alone or the ovary and oviduct are removed, as well as depending on the age of the animal (adult, cycling animals have larger ovaries with a visible corpus luteum on their surface, meaning their ovaries are easier to visualize). Similarly, many methods of hormone supplementation exist, including subcutaneous injections14, slow-release pellets15, osmotic mini pumps18, and ovarian grafting.
In this article, detailed instructions are provided on how to perform ovariectomy and prepare three types of hormone supplementation, including subcutaneous injections, slow-release pellets, and osmotic mini pumps. Two detailed protocols are provided for experimental endpoints that benefit from OVX followed by exogenous hormone supplementation (embryo transfer and artificial decidualization). This article discusses the strengths and weaknesses of each approach with the goal of guiding researchers regarding how to perform studies to isolate the impacts on the uterus, specifically in the pregnancy and fertility fields of research.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ALZET 1002 mini osmotic pumps</td>
			<td>BioScientifica</td>
			<td>1002</td>
			<td>Delivers 0.25 &#181;L/h for 14 days. Use for section 7 (Experimental procedure - Embryo transfer).</td>
		</tr>
		<tr>
			<td>ALZET 1003D mini osmotic pumps</td>
			<td>BioScientifica</td>
			<td>1003D</td>
			<td>Delivers 1 &#181;L/h for 14 days. Use for section 8 (Experimental procedure - Artificial decidualization).</td>
		</tr>
		<tr>
			<td>ALZET Reflex 7 mm clips</td>
			<td>BioScientifica</td>
			<td>0009971</td>
			<td>Either Reflex clips or Michel clips can be used for wound closure, depending on preference</td>
		</tr>
		<tr>
			<td>ALZET Reflex clip applicator</td>
			<td>BioScientifica</td>
			<td>0009974</td>
			<td>Either Reflex clips or Michel clips can be used for wound closure, depending on preference</td>
		</tr>
		<tr>
			<td>ALZET Reflex clip remover</td>
			<td>BioScientifica</td>
			<td>0009976</td>
			<td>Either Reflex clips or Michel clips can be used for wound closure, depending on preference</td>
		</tr>
		<tr>
			<td>Bupivicaine injection</td>
			<td>Pfizer</td>
			<td>NA</td>
			<td>Stock 0.5%. Use at 0.05% in saline</td>
		</tr>
		<tr>
			<td>Estradiol</td>
			<td>Sigma</td>
			<td>E8875</td>
			<td></td>
		</tr>
		<tr>
			<td>Meloxicam</td>
			<td>Ilium</td>
			<td>NA</td>
			<td>Active constituent 0.5 mg/mL. Use 3.5 mL per 200 mL cage bottle, or as your institutions vet prescribes.</td>
		</tr>
		<tr>
			<td>Michel clips</td>
			<td>Daniels</td>
			<td>NS-000242</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi purpose sealant</td>
			<td>Dow Corning</td>
			<td>732</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-surgical embryo transfer (NSET) device</td>
			<td>ParaTechs</td>
			<td>60010</td>
			<td>Contains 6 mm speculum. Single use only.</td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma</td>
			<td>P0130</td>
			<td>Soluble in ethanol. Use for&#160; section 3 (Hormone preparation - subcutaneous injection) and&#160; section 4 (Hormone preparation - slow-release pellets)</td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma</td>
			<td>P7556</td>
			<td>Soluble in water. Use for section 5 (Hormone preparation - osmotic mini pumps)</td>
		</tr>
		<tr>
			<td>Refresh eye ointment</td>
			<td>Allergan</td>
			<td>NA</td>
			<td>42.5% w/v liquid paraffin, 57.3% w/v soft white paraffin</td>
		</tr>
		<tr>
			<td>Rimadyl Carprofen</td>
			<td>Zoetis</td>
			<td>NA</td>
			<td>Stock 50 mg/mL. Use at 5 mg/kg</td>
		</tr>
		<tr>
			<td>Rubber tubing</td>
			<td>Dow Corning</td>
			<td>508-008</td>
			<td>Washed in 100% ethanol and cut into 1 cm pieces. Inside diameter 1.57 mm &#177;&#160; 0.23 mm; outside diamater 3.18 mm &#177; 0.23 mm; wall 0.81 mm.</td>
		</tr>
		<tr>
			<td>Sesame oil</td>
			<td>Sigma</td>
			<td>S3547</td>
			<td></td>
		</tr>
		<tr>
			<td>Sofsilk Silk sutures size 3-0</td>
			<td>Covidien</td>
			<td>GS-832</td>
			<td></td>
		</tr>
	</tbody>
</table>

methods for studying uterine contributions to pregnancy establishment in an ovariectomized mouse model

For pregnancy to be established, a viable blastocyst must successfully interact with a receptive uterine lining (endometrium) to facilitate implantation and placenta formation and enable ongoing pregnancy. The limitations to pregnancy success caused by embryonic defects are well known and have been largely overcome in recent decades with the rise of in vitro fertilization (IVF) and assisted reproductive technologies. As yet, however, the field has not overcome the limitations caused by an inadequately receptive endometrium, thus resulting in stagnating IVF success rates. Ovarian and endometrial functions are closely intertwined, as hormones produced by the ovary are responsible for the endometrium's menstrual cyclicity. As such, when using rodent models of pregnancy, it can be difficult to ascertain whether an observed result is due to an ovarian or uterine deficit. To overcome this, an ovariectomized mouse model was developed with embryo transfer or artificial decidualization to allow the study of uterine-specific contributions to pregnancy. This article will provide instructions on how to perform ovariectomy and offer insights into various techniques for supplying exogenous hormones to support successful artificial decidualization or pregnancy following embryo transfer from healthy donors. These techniques include subcutaneous injection, slow-release pellets, and osmotic mini pumps. The key advantages and disadvantages of each method will be discussed, enabling researchers to choose the best study design for their specific research question.

A well-characterized model of artificial decidualization is described in this protocol paper (Figure 1A). Here, young adult female mice (8 weeks old) underwent surgical ovariectomy as described in section 1 and section 2. The mice were then rested for 2 weeks to ensure that the endogenous ovarian hormones dissipated before being supported with exogenous hormones as described in sections 3-7 and section 9. Artificial decidualization was induced by an intravaginal injecti...

Watch this Scientific Journal Video about Methods for Studying Uterine Contributions to Pregnancy Establishment in an Ovariectomized Mouse Model at JoVE.com

Methods for Studying Uterine Contributions to Pregnancy Establishment in an Ovariectomized Mouse Model

Pregnancy establishment is a dynamic process involving complex embryo and uterine crosstalk. The precise contributions of the maternal uterine environment to these processes remain an active area of investigation. Here, detailed protocols are provided to aid in designing in vivo animal models to address these research questions.

For pregnancy to be established, a viable blastocyst must successfully interact with a receptive uterine lining (endometrium) to facilitate implantation ...

So it's difficult to separate the role of uterine and ovarian hormonal influences on pregnancy establishment as both are intimately linked. So this protocol allows us to directly examine the uterine contributions. While some of these techniques like ovariectomy are quite invasive, they are relatively simple to perform after practice and training, which makes them suitable for researchers that are new to mouse models.So this method can be utilized to understand the broader regulation of the HPG axis as both the ovaries and the uterus play an important role in regulation of this axis. If you've never performed these techniques before, consult with your institute's veterinarian team for training and support. To begin, apply eye lubricant generously to an anesthetized mouse by gently dabbing it on the eye after shaving a small area at dent below the spine hunch.Inject 0.1 milliliters of carprofen subcutaneously into the scruff of the neck. Test if the animal is anesthetized by pinching its back toe and checking for a reflex. Next, apply Betadine to the surgical site.Then cover it with a surgical drape. Using rat-toothed forceps, pull away the skin at the hunch upwards, and make a five millimeter longitudinal incision. Then, using blunt forceps, dissect the skin away from the underlying muscle layer down to one side toward the kidney.Identify the kidney, ovary, and ovarian fat pad through the muscle wall. Grab and lift the muscle layer with the forceps. Then, using scissors, make a 0.5 to one centimeter long incision, and pull the ovarian pad through the incision with a pair of blunt forceps.Next, using a curved needle holder, clamp underneath the ovary and oviduct at the distal end of the uterine horn and excise the ovary with scissors. After 30 seconds, remove the clamp and dab with sterile gauze. To close the muscle wall incision, lift the top of the incision using forceps.Then tie the incision with a surgeon's knot using silk sutures. Topically apply a few drops of bupivacaine using a one-milliliter syringe with no needle attachment, and dab the excess with sterile gauze. After similarly performing the ovariectomy on the other side, close the skin incision by pressing the two sides of the skin together and applying a couple of seven-millimeter surgical clips, leaving room for wound swelling.Place all required equipment on a foil inside a laminar flow or Class II biosafety hood and UV sterilize for 20 minutes. Wash the silastic tubing in 100%desinol and allow it to air dry inside the hood. Once dry, cut the tubing into one-centimeter long segments using a scalpel.Next, squeeze approximately 200 microliters of sealant into a syringe without a plunger. Replace the plunger and push out a small amount of sealant. Apply the sealant to one end of the tubing, smoothing it over with a gloved finger.And allow the sealant to dry overnight or under UV light for 30 minutes in the hood. Next, pour progesterone powder into a sterilized Petri dish. Scoop the powder into pellets with the help of forceps.Tap the sealed end of the pellet down the hood surface to condense the powder. Then seal the open end with the sealant. Wrap the Petri dish containing the pellets in foil to protect them from light.About 72 hours before subcutaneous insertion, activate the pellets by incubating them in charcoal-stripped FCS. Demonstrating this procedure will be Dr.Fiona Cousins, an early career research scientist from the Hudson Institute of Medical Research. After hormone priming the animals with subcutaneous estradiol injections, prime the animals with subcutaneous progesterone pellets two days before artificial decidualization.Before the surgery, anesthetize the mouse and test the depth of anesthesia by checking for the toe pinch reflex. Then place the mouse in a prone position, lift its tail and slowly insert a six millimeter speculum into its vagina. Place the lower body of the mouse between the first two fingers of the non-dominant hand.Then, using the index finger, gently push the tail upward to allow for better vaginal accessibility. Transfer 20 microliters of sesame oil into a non-surgical embryo transfer tip, and insert the tip through the vagina and cervix into the uterine horn. Slowly expel the oil.Once done, remove the speculum from the vagina. In this study, 80%of the the mice underwent successful decidualization, while 20%did not. So it's important to keep all your surgical equipment and working areas sterile and having patients when inserting the NSAID device through the cervix into the uterine horn of the mouse.After inducing artificial decidualization, the progesterone palate can be removed to mimic menstruation and study endometrial breakdown. Using this method, we can study uterine biology and regulation independent of the influence of the ovaries. This is really exciting in the area of fertility and women's health in general.

methods-for-studying-uterine-contributions-to-pregnancy-establishment

Research

JoVE Journal

Developmental Biology

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 ...

Development of an Ethanol-induced Fibrotic Liver Model in Zebrafish to Study Progenitor Cell-mediated Hepatocyte Regeneration

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading to cirrhosis with hepatocellular dysfunction. Among multiple hepatic insults, alcohol abuse can lead to significant health problems including liver failure and hepatocellular carcinoma. Nonetheless, the identity of endogenous cellular sources that regenerate hepatocytes in response to alcohol has not been properly investigated. Moreover, few studies have effectively modeled hepatocyte regeneration upon alcohol-induced injury. We recently reported on establishing an ethanol (EtOH)-induced fibrotic liver model in zebrafish in which hepatic progenitor cells (HPCs) gave rise to hepatocytes upon near-complete hepatocyte loss in the presence of fibrogenic stimulus. Furthermore, through chemical screens using this model, we identified multiple small molecules that enhance hepatocyte regeneration. Here we describe in detail the procedures to develop an EtOH-induced fibrotic liver model and to perform chemical screens using this model in zebrafish. This protocol will be a critical tool to delineate the molecular and cellular mechanisms of how hepatocyte regenerates in the fibrotic liver. Furthermore, these methods will facilitate potential discovery of novel therapeutic strategies for chronic liver disease in vivo.

Sustained fibrosis with deposition of excessive extracellular matrix proteins leads to cirrhosis. Alcohol abuse is one of the main causes of severe liver disease. We established an ethanol-induced zebrafish fibrotic liver model to study the mechanisms and strategies of promoting hepatocyte regeneration upon alcohol-induced injury.

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading ...

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is routinely used as an in vivo model to study embryo implantation and pregnancy progression. Unfortunately, such murine studies require pregnancy interruption to enable follow-up phenotypic analysis. To address this issue, we used three-dimensional (3-D) reconstruction of HFUS imaging data for early detection and characterization of murine embryo implantation sites and their individual developmental progression in utero. Combining HFUS imaging with 3-D reconstruction and modeling, we were able to accurately quantify embryo implantation site number as well as monitor developmental progression in pregnant C57BL6J/129S mice from 5.5 days post coitus (d.p.c.) through to 9.5 d.p.c. with the use of a transducer. Measurements included: number, location, and volume of implantation sites as well as inter-implantation site spacing; embryo viability was assessed by cardiac activity monitoring. In the immediate post-implantation period (5.5 to 8.5 d.p.c.), 3-D reconstruction of the gravid uterus in both mesh and solid overlay format enabled visual representation of the developing pregnancies within each uterine horn. As genetically engineered mice continue to be used to characterize female reproductive phenotypes derived from uterine dysfunction, this method offers a new approach to detect, quantify, and characterize early implantation events in vivo. This novel use of 3-D HFUS imaging demonstrates the ability to successfully detect, visualize, and characterize embryo-implantation sites during early murine pregnancy in a non-invasive manner. The technology offers a significant improvement over current methods, which rely on the interruption of pregnancies for gross tissue and histopathologic characterization. Here we use a video and text format to describe how to successfully perform ultrasounds of early murine pregnancy to generate reliable and reproducible data with reconstruction of the uterine form in mesh and solid 3-D images.

Mice are widely used to study gestational biology. However, pregnancy termination is required for such studies which precludes longitudinal investigations and necessitates the use of large numbers of animals. Therefore, we describe a non-invasive technique of high-frequency ultrasonography for early detection and monitoring of post-implantation events in the pregnant mouse.

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding and invasion. Current animal models for the study of T. cruzi allow limited observation of parasites in vivo, representing a challenge for understanding parasite behavior during the initial stages of infection in humans. This protozoan has a flagellar stage in both vector and mammalian hosts, but there are no studies describing its motility in vivo.The objective of this project was to establish a live vertebrate zebrafish model to evaluate T. cruzi motility in the vascular system. Transparent zebrafish larvae were injected with fluorescently labeled trypomastigotes and observed using light sheet fluorescence microscopy (LSFM), a noninvasive method to visualize live organisms with high optical resolution. The parasites could be visualized for extended periods of time due to this technique&#39;s relatively low risk of photodamage compared to confocal or epifluorescence microscopy. T. cruzi parasites were observed traveling in the circulatory system of live zebrafish in different-sized blood vessels and the yolk. They could also be seen attached to the yolk sac wall and to the atrioventricular valve despite the strong forces associated with heart contractions. LSFM of T. cruzi-inoculated zebrafish larvae is a valuable method that can be used to visualize circulating parasites and evaluate their tropism, migration patterns, and motility in the dynamic environment of the cardiovascular system of a live animal.

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

In this protocol, fluorescently labeled T. cruzi&#160;were injected into transparent zebrafish larvae, and parasite motility was observed in vivo using light sheet fluorescence microscopy.

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding ...

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

Various valved conduits and stent-mounted valves are used for right ventricular outflow tract (RVOT) valve replacement in patients with congenital heart disease. When using prosthetic materials however, these grafts are susceptible to bacterial infections and various host responses.
Identification of bacterial and host factors that play a vital role in endovascular adherence of microorganisms is of importance to better understand the pathophysiology of the onset of infections such as infective endocarditis (IE) and to develop preventive strategies. Therefore, the development of competent models to investigate bacterial adhesion under physiological shear conditions is necessary. Here, we describe the use of a newly designed in vitro perfusion chamber based on parallel plates that allows the study of bacterial adherence to different components of graft tissues such as exposed extracellular matrix, endothelial cells and inert areas. This method combined with colony-forming unit (CFU) counting is adequate to evaluate the propensity of graft materials towards bacterial adhesion under flow. Further on, the flow chamber system might be used to investigate the role of blood components in bacterial adhesion under shear conditions. We demonstrated that the source of tissue, their surface morphology and bacterial species specificity are not the major determining factors in bacterial adherence to graft tissues by using our in-house designed in vitro perfusion model.

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

We describe an in-house designed in vitro flow chamber model, which allows the investigation of bacterial adherence to graft tissues.

Various valved conduits and stent-mounted valves are used for right ventricular outflow tract (RVOT) valve replacement in patients with congenital heart ...

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 ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

An Enhanced Green Fluorescence Protein-based Assay for Studying Neurite Outgrowth in Primary Neurons

Neurite outgrowth is a fundamental event in the formation of the neural circuits during nervous system development. Severe neurite damage and synaptic dysfunction occur in various neurodegenerative diseases and age-related degeneration. Investigation of the mechanisms that regulate neurite outgrowth would not only shed valuable light on brain developmental processes but also on such neurological disorders. Due to the low transfection efficiency, it is currently challenging to study the effect of a specific protein on neurite outgrowth in primary mammalian neurons. Here, we describe a simple method for the investigation of neurite outgrowth by the co-transfection of primary rat cortical neurons with EGFP and a protein of interest (POI). This method allows the identification of POI transfected neurons through the EGFP signal, and thus the effect of the POI on neurite outgrowth can be determined precisely. This EGFP-based assay provides a convenient approach for the investigation of pathways regulating neurite outgrowth.

In this report, we describe a simple protocol for studying neurite outgrowth in embryonic rat cortical neurons by co-transfecting with EGFP and the protein of interest.

Neurite outgrowth is a fundamental event in the formation of the neural circuits during nervous system development. Severe neurite damage and synaptic ...

Analysis of Congenital Heart Defects in Mouse Embryos Using Qualitative and Quantitative Histological Methods

Congenital heart defects (CHD) are the most common type of birth defect in humans, affecting up to 1% of all live births. However, the underlying causes for CHD are still poorly understood. The developing mouse constitutes a valuable model for the study of CHD, because cardiac developmental programs between mice and humans are highly conserved. The protocol describes in detail how to produce mouse embryos of the desired gestational stage, methods to isolate and preserve the heart for downstream processing, quantitative methods to identify common types of CHD by histology (e.g., ventricular septal defects, atrial septal defects, patent ductus arteriosus), and quantitative histomorphometry methods to measure common muscular compaction phenotypes. These methods articulate all the steps involved in sample preparation, collection, and analysis, allowing scientists to correctly and reproducibly measure CHD.

In this protocol, we describe procedures to qualitatively and quantitatively analyze developmental phenotypes in mice associated with congenital heart defects.

Congenital heart defects (CHD) are the most common type of birth defect in humans, affecting up to 1% of all live births. However, the underlying causes ...

An Optimized O9-1/Hydrogel System for Studying Mechanical Signals in Neural Crest Cells

Neural crest cells (NCCs) are vertebrate embryonic multipotent cells that can migrate and differentiate into a wide array of cell types that give rise to various organs and tissues. Tissue stiffness produces mechanical force, a physical cue that plays a critical role in NCC differentiation; however, the mechanism remains unclear. The method described here provides detailed information for the optimized generation of polyacrylamide hydrogels of varying stiffness, the accurate measurement of such stiffness, and the evaluation of the impact of mechanical signals in O9-1 cells, a NCC line that mimics in vivo NCCs.
Hydrogel stiffness was measured using atomic force microscopy (AFM) and indicated different stiffness levels accordingly. O9-1 NCCs cultured on hydrogels of varying stiffness showed different cell morphology and gene expression of stress fibers, which indicated varying biological effects caused by mechanical signal changes. Moreover, this established that varying the hydrogel stiffness resulted in an efficient in vitro system to manipulate mechanical signaling by altering gel stiffness and analyzing the molecular and genetic regulation in NCCs. O9-1 NCCs can differentiate into a wide range of cell types under the influence of the corresponding differentiation media, and it is convenient to manipulate chemical signals in vitro. Therefore, this in vitro system is a powerful tool to study the role of mechanical signaling in NCCs and its interaction with chemical signals, which will help researchers better understand the molecular and genetic mechanisms of neural crest development and diseases.

Detailed step-by-step protocols are described here for studying mechanical signals in vitro using multipotent O9-1 neural crest cells and polyacrylamide hydrogels of varying stiffness.

Neural crest cells (NCCs) are vertebrate embryonic multipotent cells that can migrate and differentiate into a wide array of cell types that give rise to ...