The overall goal of this procedure is to grow neonatal ovaries and or individual ovarian follicles in vitro. This is accomplished by first dissecting ovaries from cold females of the appropriate age. The second step is to prepare the tissue, including fine dissection of individual ovarian follicles with acupuncture needles.
Then the neonatal ovaries or individual pre antral follicles are cultured with regular feeding, and the final step is to fix or freeze the tissue for histological or molecular analysis. Ultimately, the culture system supports development in a highly physiological manner and can be used to examine the regulation of ovarian follicle development and to investigate interactions between primordial and growing ovarian follicles. The main advantage of this technique is that intact ovarian follicles are supported through follicle development, maintaining their three-dimensional structure without the need of supporting matrices.
This method can help answer key questions in the ovarian biology field, and also to help investigate the effects of toxic compounds such as chemotherapy, drugs on the ovary, and hence female fertility. We first had the idea for co culturing different ages and hence stages of ovarian follicles when discussing how to investigate the effects of growing follicles on the resting primordial follicle pool, Demonstrating the technique alongside Steph Morgan and Lisa Campbell will be Vivian Allison, a technician from the laboratory and Allison Murray, who was a postdoctoral scientist in the lab for many years. Both of them helped develop key aspects of the technique To pull the pipettes for this protocol.
Heat and bend glass pipettes over a flame and make a clean cut using a glass cutter. Then sterilize the curved glass at 160 degrees Celsius for about 45 minutes. It takes practice to get the right curve in the pipette for your technique.
Always cut the pipette with a glass cutter so there's no jagged glass. You also want to ensure that you have the end up with the right diameter for your tissue Built to sterilize the dissection medium made of lebowitz L 15 medium with BSA through a 0.2 micron filter. That is 25 millimeters in diameter built to sterilize the neonatal ovary culture.
Medium through another 0.2 micron filter just 25 millimeters in diameter and collect it in a sterile tube. Then add one milliliter of the neonatal medium to each well of a 24. Well plate and transfer a membrane onto the surface of the medium in each well.
The follicle culture medium is also filter sterilized like the ovary culture medium. Then silicon oil must be filter sterilized as well. Next, repair 96 well culture plates with 30 microliter drops of the follicle medium along the top row.
Carefully overlay these wells with 70 microliters of silicon oil. Alternatively, follicle follicle co-culture requires 100 microliters of medium with an overlay of 100 microliters of silicon oil. Then prepare follicle ovary co-culture plates with one milliliter of medium per well and a very carefully placed UV sterilized nuclear po membrane.
Begin by transferring one milliliter of dissection medium into each sterile glass embryo dish. After coloring the zero to five postnatal day old pups, grasp the skin covering the abdominal wall with fine forceps and inci the skin and body wall. Pull open this large incision exposing the abdomen.
The bladder is usually engorged at this stage and can be punctured to make dissection easier. Using watchmaker forceps, move aside the guts. Then on either side, follow the uterine horns from the bladder up to the kidney.
The ovary is a cloud-like structure located just below the kidney at the top of the uterus. Grasp the ovary gently and using scissors. Cut it free from the uterus.
After dissecting out both ovaries, transfer them to dishes filled with warm dissection.Medium. Use a dissection microscope with a stage heated at 37 degrees Celsius to dissect the ovaries using insulin needles. Trim away the basal sack and any excess material, including the fallopian tube until only the ovary remains.
Then transfer each ovary into the well of a prepared plate using a curved pipette. Place one ovary on each membrane culture, the tissue changing half the medium every other day for up to six days when changing the medium place the pipette tip at the edge of the well of medium to avoid disturbing the membrane. After the culturing, the ovaries can be stored frozen or fixed or histology or immunochemistry.
After collecting the ovaries from older mice in a dish of warm dissection medium, remove the ovarian Basa. Use insulin needles to trim away the basal sack and any excess material, including the fallopian tube until only the ovary remains. Then cut the ovaries in half using the needles and carefully transfer each half into an individual watch glass containing one milliliter of dissection medium.
Cover the watch glasses with a glass slide and store them in the oven for up to an hour as soon as possible. Working under a microscope with a heated stage in a lamina flow hood, dissect the ovaries into large pieces using insulin needles to identify late pre antal follicles. The pre antal follicles contain two to three layers of granulosis cells and have a diameter of around 180 to 200 micrometers.
Dissect any of these out using an inline needle and an acupuncture needle secured by a needle holder, avoid damaging the basal lamina, but be sure to remove the surrounding S stroma. This takes practice. The follicle needs some fecal tissue, but not too much.
Also, if the dissection process is too rough, the follicle become damaged and either rupture or die. During culture, User prepared perpe to carefully transfer the follicles to a new medium filled watch glass. Keep the follicles on the thin caliber section of the perpet using a calibrated eyepiece fitted to a dissecting microscope.
Measure the follicles. If they're within 10 micrometers of a 190 micrometer diameter. They might be used for culturing, discard the other follicles from the selected follicles.
Choose only the healthiest for culturing. They should be translucent without darker retic areas have an intact basal laminar and some attached thecal tissue. A good yield is 10 to 15 follicles per ovary.
Now using a prepared curved perpe, transfer these follicles to the prepared 96 well culture plate. Place one follicle in each well below the oil layer and culture. The follicles are up to six days every day.
Prepare a new row with media. An hour or more later, transfer the follicles to the next row of fresh equilibrated media or follicle follicle co cultures culture two follicles side by side in one. Well instead of transfers, use a fine gel tip to change half the medium every other day.
For follicle ovary, co cultures transfer a follicle to a neonatal ovary placed on a membrane in a culture plate, carefully put the follicle beside one pole of the neonatal ovary so that they can then be moved into contact with each other. Maintain this culture for up to five days. Replacing 500 microliters of the medium every other day.
The pre antal follicles often become encapsulated by the neonatal ovaries. During this culture, neonatal ovaries from newborn were cultured for six days and exposed to reproductive toxicants. On the second day of culture, the tissues were processed to determine the number and health of different stages of follicles.
One investigated toxicant was cisplatin. The other toxicant investigated was doxorubicin. A different experiment investigated the effect of gonadotropin follicle stimulating hormone and luteinizing hormone on the growth of individual follicles.
But this analysis follicle size was periodically measured to map their growth. Follicle follicle co-culture was investigated using A GFP expressing follicle and a wild type follicle processes extend from one to the other. Immunohistochemical processing of this co-culture revealed that the GFP processes lay within the wild types.
Thecal layer, wild type neonatal ovaries were also co-culture with the GFP expressing follicle. In this scenario, the GFP processes migrated through the ovarian interstitium. The neonatal ovary culture is quite a straightforward method, but expect to spend some time mastering the technique of dissecting undamaged intact pre antral follicles.
This is essential to their proper development. Following the follicle culture, oocytes can be fertilized by in vitro fertilization, after which embryos can be transferred into pseudo pregnant females and live young obtained.
