We would like to thank members of the gynaecology oncology group for critical reading of this manuscript. This work is in part supported by funding from the National Health and Medical Research Council, the Australian Research Council, and the Cancer Institute NSW (P.S.T). M.K. is a recipient of the University of Newcastle Postgraduate Research Fellowship.

Animal care and experimental procedures were conducted in accordance with the guidelines of the Animal Care and Ethics Committee of the University of Newcastle and confirmed to the New South Wales Animal Research Act, New South Wales Animal Research Regulation, and the Australian code for the care and use of animals for scientific purposes. All the procedures undertaken on mice were approved by the Animal Care and Ethics Committee of the University of Newcastle.
1. Time Mating
<ol>
	<li>Pair...

<ol>
	<li>Joseph, A., Yao, H., Hinton, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+morphogenesis+of+the+Wolffian/epididymal+duct,+more+twists+and+turns.">Development and morphogenesis of the Wolffian/epididymal duct, more twists and turns.</a> Dev Biol. 325, 6-14 (2009).</li><li>Murashima, A., Xu, B., Hinton, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+normal+and+abnormal+development+of+the+Wolffian/epididymal+duct+by+using+transgenic+mice.">Understanding normal and abnormal development of the Wolffian/epididymal duct by using transgenic mice.</a> Asian J Androl. 17, 1-7 (2015).</li><li>Carroll, T. J., Park, J. S., Hayashi, S., Majumdar, A., McMahon, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt9b+plays+a+central+role+in+the+regulation+of+mesenchymal+to+epithelial+transitions+underlying+organogenesis+of+the+mammalian+urogenital+system.">Wnt9b plays a central role in the regulation of mesenchymal to epithelial transitions underlying organogenesis of the mammalian urogenital system.</a> Dev Cell. 9, 283-292 (2005).</li><li>Kumar, M., Syed, S. M., Taketo, M. M., Tanwar, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+Wnt/&#946;catenin+signalling+is+essential+for+epididymal+coiling.">Epithelial Wnt/&#946;catenin signalling is essential for epididymal coiling.</a> Dev Biol. 412, 234-249 (2016).</li><li>Murashima, A., 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=Essential+roles+of+androgen+signaling+in+Wolffian+duct+stabilization+and+epididymal+cell+differentiation.">Essential roles of androgen signaling in Wolffian duct stabilization and epididymal cell differentiation.</a> Endocrinology. 152, 1640-1651 (2011).</li><li>Lin, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+and+limitations+of+genetically+modified+mouse+models+in+drug+discovery+and+development.">Applications and limitations of genetically modified mouse models in drug discovery and development.</a> Curr Drug Metab. 9, 419-438 (2008).</li><li>Rak-Raszewska, A., Hauser, P. V., Vainio, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+In+Vitro+Culture:+What+Have+We+Learned+about+Early+Kidney+Development?.">Organ In Vitro Culture: What Have We Learned about Early Kidney Development?.</a> Stem Cells Int. 959807, 1-16 (2015).</li><li>Hirashima, T., Adachi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Procedures+for+the+Quantification+of+Whole-Tissue+Immunofluorescence+Images+Obtained+at+Single-Cell+Resolution+during+Murine+Tubular+Organ+Development.">Procedures for the Quantification of Whole-Tissue Immunofluorescence Images Obtained at Single-Cell Resolution during Murine Tubular Organ Development.</a> PLoS One. 10, 0135343 (2015).</li><li>Chan, W. Y., Blomberg, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germline+Development:+Methods+and+Protocols.">Germline Development: Methods and Protocols.</a> Methods Mol Biol. 825, (2012).</li><li>Karner, C. M., 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=Tankyrase+Is+Necessary+for+Canonical+Wnt+Signaling+During+Kidney+Development.">Tankyrase Is Necessary for Canonical Wnt Signaling During Kidney Development.</a> Dev Dyn. 239, 2014-2023 (2010).</li><li>Traykova-Brauch, M., 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=An+efficient+and+versatile+system+for+acute+and+chronic+modulation+of+renal+tubular+function+in+transgenic+mice.">An efficient and versatile system for acute and chronic modulation of renal tubular function in transgenic mice.</a> Nat Med. 14, 979-984 (2008).</li></ol>

The organ culture system has many advantages over the traditional cell culture system. This system retains the original structural relationships and interactions between different cell types. It mimics in vivo systems and gives more accurate information than cell culture studies where the niche factor is absent. The use of organ culture systems even have advantages over using the whole animal. These include the higher cost of using the whole animal, easy care and maintenance of organ culture system, etc.</em...

The male reproductive system primarily consists of testis, the site for germ cell development and differentiation, and a complex ductal system that is required for the maturation, transport, and storage of sperm. Epididymis is a tubular organ that connects testis with vas deferens and mainly involved in post-testicular development and maturation of germ cells 1. The highly coiled adult mouse epididymis develops from a simple and straight precursor tube, Wolffian duct (WD) 1. The complex and coiled structure of epididymis is essential for sperm to acquire the capacity to fertilize female germ cells 2. How such an essential organ for male fertility develops and gets into shape is not well understood. Various signaling pathways have been shown to be involved in the development of WD such as the Wnt signaling pathway 3,4 and the androgen signaling pathway 5. Our recent work has established the requirement of balanced Wnt signaling&#160;for WD coiling during prenatal development 4. However, further studies are required to understand the mechanisms involved in postnatal epididymal coiling, cellular differentiation, and cell-to-cell communication within the epididymal epithelium and with interstitial cells.
Genetically modified mouse models have been proven to be a powerful tool for identification/validation of molecular mechanisms underlying development and disease of various organ systems 6. Despite its extensive use, there are significant limitations of genetically modified mouse models, including the unpredicted phenotype of targeted mouse mutants with regards to presumed gene function, and no phenotype in null mutants in some models, influence of genetic and environmental factors, labor intensive and time-consuming process of developing mouse models. Therefore, the interpretation of the significance of the findings only from genetically modified mouse models is not always straightforward 6. These limitations can be overcome by using in vitro organ culture system which gives us the flexibility to manipulate multiple signaling pathways in real time in controlled culture conditions. Organ culture system is instrumental in understanding the physiology and pathology of whole organs 7. Moreover, imaging of whole organs stained with fluorophore labeled antibodies enables us to visualize the markers of interest in a three-dimensional context, as it exists in vivo, thereby providing a better understanding of the organ&#39;s shape, structure, and function 8.
Here, we have described the methods for isolation of mouse embryonic gonadal ridges, in vitro culture&#160;and whole mount immunofluorescence imaging of WDs, that can be applied to answer a variety of questions concerning the morphogenesis of tubular organs such as WDs. In this protocol, we isolated mouse embryonic urogenital ridges from 15.5 days post coitum (dpc) pregnant dams and cultured them for 3 d&#160;followed by immunostaining for an epithelial cell marker (cytokeratin 8, CK8), a cell proliferation marker (phospho-Histone 3, PH3) and active &#946;catenin.

<table border="0" cellpadding="0" cellspacing="0" width="769">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">24 well plates</td>
			<td style="width:195px;">Corning, USA</td>
			<td style="width:136px;">3524</td>
			<td style="width:191px;">can be purchased from other vendors</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Hank&#8217;s Balanced Salt Solution (HBSS)</td>
			<td style="width:195px;">GE Healthcare Life Sciences, USA</td>
			<td style="width:136px;">SH30031.02</td>
			<td style="width:191px;">can be purchased from other vendors</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Dulbecco&#39;s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12)</td>
			<td style="width:195px;">Sigma, USA</td>
			<td style="width:136px;">D8437</td>
			<td style="width:191px;">Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:195px;">Interpath,&#160; Australia</td>
			<td style="width:136px;">SH30034.02</td>
			<td style="width:191px;">10% FBS used in culture medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">L-Glutamine</td>
			<td style="width:195px;">GE Healthcare Life Sciences, USA</td>
			<td style="width:136px;">SH30034.01</td>
			<td style="width:191px;">1% concentration used</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:247px;">Penicillin-Streptomycin</td>
			<td style="width:195px;">Gibco, USA</td>
			<td style="width:136px;">15070-063</td>
			<td style="width:191px;">1% concentration used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Whatman Nuclepore Polycarbonate Track-Etch&#160;</td>
			<td style="width:195px;">Whatman, USA</td>
			<td style="width:136px;">110409</td>
			<td style="width:191px;">Membrane (0.8 mm)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">IWR1</td>
			<td style="width:195px;">Sigma, USA</td>
			<td style="width:136px;">10161-5mg</td>
			<td style="width:191px;">100 &#181;M concentration used</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:247px;">Paraformaldehyde (PFA)</td>
			<td style="width:195px;">Electron Microscopy Sciences, USA</td>
			<td style="width:136px;">15710</td>
			<td style="width:191px;">16% stock, 4% in PBS for use. TOXIC - wear gloves and cannot 
			be disposed of in the sink</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Ethanol</td>
			<td style="width:195px;">Thermo Scientific Fisher, USA</td>
			<td style="width:136px;">214-20L PL</td>
			<td style="width:191px;">Absolute, make 25, 50 and 75% with milliQ water.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Tween-20</td>
			<td style="width:195px;">Thermo Scientific Fisher, USA</td>
			<td style="width:136px;">2509-500ml</td>
			<td style="width:191px;">Very viscous, careful while dispensing</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Triton X-100</td>
			<td style="width:195px;">Sigma, USA</td>
			<td style="width:136px;">T8787-50ml</td>
			<td style="width:191px;">Very viscous, careful while dispensing</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Di-Sodium Hydrogen Phosphate</td>
			<td style="width:195px;">Thermo Scientific Fisher, USA</td>
			<td style="width:136px;">621-500mg</td>
			<td style="width:191px;">can be purchased from other vendors</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Sodium Di-hydrogen Phosphate</td>
			<td style="width:195px;">Ajax Finechem, USA</td>
			<td style="width:136px;">4745-500g</td>
			<td style="width:191px;">can be purchased from other vendors</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Sodium Chloride</td>
			<td style="width:195px;">Thermo Scientific Fisher, USA</td>
			<td style="width:136px;">465-500g</td>
			<td style="width:191px;">can be purchased from other vendors</td>
		</tr>
		<tr height="83">
			<td height="83" style="height:83px;width:247px;">Cytokeratin8/Troma I</td>
			<td style="width:195px;">Developmental Studies Hybridoma Bank, USA (DHSB)</td>
			<td style="width:136px;">TROMA-I</td>
			<td style="width:191px;">1:250 in blocking buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">active &#946;catenin&#160;</td>
			<td style="width:195px;">Cell Signalling Technology,&#160; USA</td>
			<td style="width:136px;">D13A1</td>
			<td style="width:191px;">1:200 in blocking buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;"></td>
			<td style="width:195px;"></td>
			<td style="width:136px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:247px;">Phospho-Histone3</td>
			<td style="width:195px;">Millipore, MA, USA</td>
			<td style="width:136px;">06-570</td>
			<td style="width:191px;">1:200 in blocking buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Alexa488 Goat anti-rabbit IgG</td>
			<td style="width:195px;">Jackson ImmunoResearchLabs,&#160; USA</td>
			<td style="width:136px;">111-545-047</td>
			<td style="width:191px;">1:250 in blocking buffer</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:247px;">Alexa594 Goat anti-Rat IgG</td>
			<td style="width:195px;">Jackson ImmunoResearchLabs,&#160; USA</td>
			<td style="width:136px;">112-585-072</td>
			<td style="width:191px;">1:250 in blocking buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Glycerol</td>
			<td style="width:195px;">Thermo Fisher Scientific, USA</td>
			<td style="width:136px;">242-500ml</td>
			<td style="width:191px;">can be purchased from other vendors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">n-Propyl galate</td>
			<td style="width:195px;">Sigma, USA</td>
			<td style="width:136px;">2370</td>
			<td>-</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">2-(4-amidinophenyl)-1H -indole-6-carboxamidine (DAPI)</td>
			<td style="width:195px;">Sigma, USA</td>
			<td style="width:136px;">D9564-10mg</td>
			<td style="width:191px;">Use to stain nucleic acids (DNA)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:195px;">Thermo Fisher Scientific, USA</td>
			<td style="width:136px;">2225-500ml</td>
			<td style="width:191px;">Solvent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Cover Slips (24x50 mm)</td>
			<td style="width:195px;">Lomb Scientific</td>
			<td style="width:136px;">CS24501GP</td>
			<td>-</td>
		</tr>
	</tbody>
</table>

organ culture and whole mount immunofluorescence staining of mouse wolffian ducts

Tubal morphogenesis is a fundamental requirement for the development of most mammalian organs, including the male reproductive system. The epididymis, an integral part of the male reproductive tract, is responsible for sperm storage, maturation, and transport. The adult epididymis is a highly coiled tube that develops from a simple and straight embryonic precursor known as Wolffian duct (WD). Proper coiling of the epididymis is essential for male fertility, as sperm in the testis are unable to fertilize an oocyte. However, the mechanism responsible for epididymal development and coiling remains unclear, partially due to the lack of whole organ culture and imaging methods. In this study, we describe an in vitro culture system and whole mount immunofluorescence protocol to better visualize the process of WD coiling and development, which may also be applied to study other tubular organs.

During development, the WD undergoes significant changes wherein a simple and straight tube is transformed into a highly complex and coiled duct. Using methods described above, WDs undergo a similar transformation in culture conditions. Here we have shown the results from WDs cultured for 3 d&#160;(Figure 3). To dissect molecular mechanisms involved in WD morphogenesis, the culture medium can be supplemented with inhibitors and activators targeting different signaling pathways. Figure 3C...

Watch this Scientific Journal Video about Organ Culture and Whole Mount Immunofluorescence Staining of Mouse Wolffian Ducts at JoVE.com

Organ Culture and Whole Mount Immunofluorescence Staining of Mouse Wolffian Ducts

We present a method for isolation and culture of the mouse Wolffian duct (WD). We also demonstrate a detailed procedure for whole mount immunostaining of cultured/freshly isolated WDs with fluorescently tagged antibodies. Together, these techniques enable the study of WD development, coiling, and differentiation.

Tubal morphogenesis is a fundamental requirement for the development of most mammalian organs, including the male reproductive system. The epididymis, an ...

The overall goal of this organ culture and whole mount immunofluorescene method is to better visualize and understand the process of Wolffian induct coiling and development. This method will help to answer key questions in the field of developmental biology about the mechanisms involved in development of organ shape, structure, and function. The main advantages of this technique are that it mimics in vivo systems and provides more accuracte information than cell culture studies which lack niche factors.Before noon on day 15 post-conception, place a pregnant mouse in the supine position on absorbent tissue paper, and spray the ventral side of the abdomen with 70%ethanol. Using forceps, lift the lower abdominal skin on the ventral side of the animal, and use surgical scissors to make a lateral incision extending from the urogenital opening to the rib cage. Grasp the urinary bladder with the forceps just above the cervix, and make an incision into the uterus.Holding the urinary bladder, make an incision into the uterine broad ligament, and utero tubal junction to detach the uterus from paratenigal attachment. Place the uterus in a 50 milliliter tube containing ice cold HBSS, and gently agitate the tissue to remove the excess blood. Transfer the rinsed uterus to a Petri dish containing fresh ice cold HBSS on ice, and cut the uterine wall to remove the embryos, placing them in a new Petri dish with fresh HBSS on ice as they are harvested.Place an embryo on its lateral side onto an ethanol-soaked sterile piece of tissue paper in a new dish. And use a sterile blade to make a diagonal incision across the lower abdomen of the animal. Pin the embryo on a sterile sponge base along the vertebral column, and move the embryo under a dissecting stereo microscope.Carefully cut along the ventral mid-line and remove the liver and intestines. The urogenital system should now be visible. Make an incision in the vas deferens, close it its urethral attachment.Followed by an incision thought the attachment of the lower portion of the Wolffian duct to the gubernaculum to harvest the testes and Wolffian ducts. Placing the tissues in a new Petri dish with fresh HBSS on ice as they are collected. To culture the embryonic gonadal ridges, first add 300 microliters of medium per well to a 24-well cell culture plate.Next, add a small drop of sterile HBSS into a new Petri dish and place a 0.8 micrometer poly carbonate track etch membrane onto the drop with the shining surface facing the HBSS. Always place the membrane on the top of the drop of HBSS to keep the tissue hydrated with the rough side of the membrane facing up. Using clean forceps, place two genital ridges onto the rough side of the membrane without the tissues touching each other.And use a sterile wipe to remove the excess HBSS. While you're transferring the genital ridges onto the membrane, pick up the sample between the two arms of forceps, taking care to remove any excess HBSS from the membrane after the transfer to prevent cystic growth of Wolffian ducts. Then place the membrane into one well of the 24-well plate to culture the tissues of the ear medium interface at 37 degrees Celsius, and 5%carbon dioxide, with a total medium change every day for three days.By the fourth day of culture, the uncoiled Wolffian ducts will have transformed into highly convoluted tubes. To fix the tissues for whole mount immunofluorescene, transfer the membranes with the cultured gonads and Wolffian ducts into new Petri dishes containing ice cold PBS. The membranes will float on the PBS.Use the forceps tip to sink the membranes. And transfer the cultured gonadal tissues into 4%paraformaldehyde. After fixation, rinse the samples with three 10-minute washes in in PBS plus Triton X with gentle rocking at room temperature.Then dehydrate the tissues in a graded ethanol series for 10 minutes for each immersion at four degrees Celsius and 30 rotations per minute. To switch the samples between immersions, allow the tissues to settle during the last two minutes of the dehydration. Then remove as much alcohol as possible without touching the tissues, and add the next higher concentration of ethanol.After the last dehydration, rehydrate the tissues in a reverse-graded ethanol series as just demonstrated. Then wash the tissues in PBS plus Triton X four times for 20 minutes at room temperature. And 30 rotations per minute for each wash.Block the tissues for one hour at room temperature with blotting buffer, followed by an overnight incubation at four degrees Celsius and 30 rotations per minute in the primary antibody of interest. The next day wash the tissues four times in PBS plus nonfat milk powder in tween for 30 minutes at room temperature, and 30 rotations per minute for each wash. After the last wash, label the tissues with the appropriate secondary antibody for one hour at room temperature, with slow rocking and protected from light.Then wash the tissues three times in PBS plus tween only. And analyze the samples by immunofluorescene microscopy. During development, the Wolffian duct undergoes significant changes, wherein a simple and straight tube is transformed into a highly complex and coiled duct.To dissect the molecular mechanisms involved in Wolffian duct morphogenesis, the culture medium can be supplemented with inhibitors and activators that target different signaling pathways. For example, this Wolffian duct remained uncoiled after three days of culture in the presence of a one to signaling specific inhibitor. A pathway apparently involved in coiling.Whole mount immunostaining of Wolffian ducts reveals the expression of cytokeratin-8, and the presence of PH3 positive proliferating cells and three-day culture tissues, as well as the expression of beta catenin in freshly-isolated day 18.5 post-conception whole mounted tissues. Once mastered, this technique can be completed in about five to 10 minutes per embryo if performed properly. By following this procedure, pharmacological agents or hormones can be tested on the culture organs to answer additional questions about the direct effect of these agents on specific gonadal tissues.While attempting this procedure, it is important to remember not to grasp the genital ridges directly with the forceps. After its development, this technique paved the way for researchers in the field of developmental biology to explore the mechanisms involved in congenital abnormalities and anomalies development of the reproductive system. After watching this video, you should have a good understanding of how to culture gentile ridges and perform whole-mount immunofluorescene.Don't forget that working with paraformaldehyde can be extremely hazardous, and that personal protective equipment should always be worn while handling this material.

organ-culture-whole-mount-immunofluorescence-staining-mouse-wolffian

Research

JoVE Journal

Developmental Biology

10.6K vistas.  University of Newcastle. The overall goal of this organ culture and whole mount immunofluorescene method is to better visualize and understand the process of Wolffian induct coiling and development. This method will help to answer key questions in the field of developmental biology about the mechanisms involved in development of organ shape, structure, and function. The main advantages of this technique are that it mimics in vivo systems and provides more accuracte information than cell culture studies which lack nic...