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.
