Kathryn Polkoff Research Summary Abstract A comparison of two embedding systems for follicle culture Recent research has focused on developing secondary, pre-antral follicles as a new way to collect female gametes to develop in vitro or to study folliculogenesis. Secondary follicles are abundant in ovaries of almost all females, and can be isolated from the ovarian cortex of fresh or cryopreserved tissue. These follicles must be matured to obtain mature oocytes, but the mechanism for early follicle development is not well understood. Currently, culture techniques are being developed to promote the growth and quality of these follicles. The aim of this preliminary study was to compare two embedding systems for porcine follicles to improve preantral follicle culture. We collected secondary pre-antral follicles isolated from porcine ovaries recovered from the abattoir. Ovaries were transported to our laboratory in saline solution (0.9% NaCl) and cut into smaller fragments with a scalpel. Follicles were further mechanically isolated with a 23 ½ gage needle and placed into a dish with medium TCM199 plus Hepes (Lonza 12117F) supplemented with 5% fetal bovine serum (FBS). Secondary follicles less than 300 µm were selected. Furthermore, only clear follicles with dark oocytes and no antrum were used. The selected follicles were randomly divided into 5 different treatment groups: 1) embedded in 0.5% alginate gel; 2) 1.0% alginate gel; 3) 1.0% agarose gel; 4) 2.0% agarose gel, 5) control. Three follicles were placed in each well with 280 µL α-MEM medium supplemented with 3.5 μg/ml insulin 10 μg/ml transferrin, 100 μg/ml L-ascorbic acid, 7.5% porcine serum and cultured for 4 days at 39C at 5% CO2. Media (140µL) from each well was changed on day 2 and saved for metabolic and functional analysis. Initial diameters and measurements on day 2 and day 4 were taken to analyze dimensional growth. We evaluated 6 follicles per group per replicate, for a total of 3 replicates (18 per group). All recorded parameters were subjected to a two way analysis of variance using the procedure of the Generalized Linear Model (SPSS, 18th version, 2009). Independent variable was the result of the balance between the size on day 2 and day 0 and the size on day 4 and day 0 time of observation. Data were normally distributed. Least square means tests were used to perform statistical multiple comparisons. The alpha level was set at 0.05. All data were expressed as quadratic means and mean standard errors. The results in the Table 1 show that at day 2 there was no difference between groups. However, after 4 day of development the group Agar 2% and Alginate 1% showed a statistical difference when compared with the control. These results suggest that there is a positive effect when the follicles are embedded during culture. Table 1. Follicle growth (in µm) since day 0 for each treatment. Day 2 Day 4 a,b Control 23.4 ( 38.1) 11.2 (39.5) a Alginate 0.5% 31.4 (43.1) 35.7 (44.7) Alginate 1% 42.4 (36.8) 77.9 (17.6) b Agar 1% 3.8 (118.8) 40.0 (89.3) Least square means (± SD) within each row without common superscripts differ (P< 0.05). Summary Agar 2% 29.9 (45.4) 76.2 (48.6) b The intention of this research was to examine methods of culturing follicles and come up with a system that could improve their growth and quality. Follicles are found in the ovaries of both mature and immature females, and they contain oocytes. At birth, the ovaries contain all of the oocytes they will ever have, and are not capable of generating new ones. Before maturation, follicles are classified as primordial, they are very small and their contained oocytes are not developed enough for fertilization. As a series of factors act upon these follicles, of which the exact combination of specifics is unknown, they begin to progress into primary then secondary follicles, growing in size and changing slightly with respect to cell type and cell structure. They then develop a larger cavity, called an antrum, which is a fluid filled space, and when the female ready to ovulate, these follicles rupture and emit a mature ovum. The follicles I used for this project were secondary follicles before they developed an antrum. The aim of this research was to see if putting secondary, pre-antral follicles into a gel matrix while they are being cultured helps in their growth and development. The idea behind this is that the gel would mimic the structure of the ovary and that the pressure on the follicles would be more similar to the in vivo environment. To test this, we began with porcine ovaries from the slaughterhouse. Ovaries were cut into smaller fragments by slicing off thin strips of the cortex (the outer part of the ovary where the follicles can be found) with a scalpel. We then mechanically isolated the follicles under a microscope using needles, specifically selecting follicles 200-300 µm in size and with no visible antrum. Once the follicles were isolated and selected, they were randomly divided into treatment groups. The four different treatments used were of varying gel concentration and substances.. We used 0.5% and 1% solutions of Alginate, and 1% and 2% solutions of Agarose. We made these gels by heating up deionized water and adding the desired concentration of solutes. To make sure there were no impurities that could affect the growth of the follicles, we added activated charcoal (which binds to impurities) and filtered the solution (which removes the charcoal and other impurities). While the solutions were still warm in a liquid state, we used a pipette to transfer the follicles into a drop of the gel. Once the follicles were coated, they were sucked back up into the pipette and allowed to cool slightly so that the gel would cool and solidify around the follicles, and then they were placed in a culture well for four days with three follicles in each well. Each well contained media, which is a solution of important nutrients for the growth and development of the follicles, and was incubated at 39C, which is a similar temperature to the body temperature of the pig. After two days of culture, half of the media was removed (and saved as a sample) and replaced with new media. We measured the growth of the follicles by taking pictures with a microscope and measuring the average diameter on the initial day, day two, and day four of culture. I repeated this process four times, isolating over forty follicles each replicate. As you can see from the table in the abstract above, we found that the follicles had increased growth when they were cultured embedded in gel than when they were free in culture. At the conclusion of the summer, I submitted the above abstract to International Embryo Transfer Society (IETS). The abstract has been accepted and I will be presenting a poster on this research in January. This fall I will continue working on varying aspects of this project. First, I will complete more replicates of the experiment so that I can gather more data in order to publish a paper on this project. Furthermore, I will analyze development of the follicles based on not only morphological growth, but I will also evaluate the hormones and steroids produced by follicles in each system based on the samples of media at day 2 and day 4 and check that they are being produced in proper ratios. I will also use a polymerase chain reaction (PCR) to check for IGF-I, which is indicative of growth and also check for sirtuin, which is indicative of cell death. It is important to study follicle culture for several reasons. First of all, in reproduction, the limiting factor for the amount of embryos that can be made is the number of female gametes available. With access to all types of follicles instead of just the ones ready to ovulate, there are hundreds of oocytes at our finger tips. If we are interested in breeding a certain line of livestock, we do not have to wait for offspring to reach sexual maturity before we use their gametes; instead we can collect follicles and culture them in vitro. In another instance, women facing a cancer diagnoses can have their ovaries removed and preserved for a long time through cryopreservation (freezing) to avoid the problems and damages to the ovary associated with chemotherapy or radiation therapy. The cryopreserved tissue can then be used to culture follicles in vitro and produce mature oocytes, meaning that even young cancer patients can have their natural children after recovering from cancer without worrying about reintroducing cancer cells into their body. During this summer, thanks to the summer undergraduate research fellowship, I was also able to be a part of several other projects in my lab that I worked on concurrently with my follicle embedding project. I finished collecting data from a previous follicle research project experimenting with adding hormones to the media, to contribute to the research started by two PhD students who had to return to Brazil for their defenses. I contributed to a colleague’s abstract by testing embryos and semen for Bovine Leukosis Virus, which is a virus very common in dairy cattle, the findings of which research will be presented in January. I also contributed data to another abstract examining the embryo metabolism for female versus male bovine embryo. Finally, I was able to participate in several surgeries focusing on regenerative medicine using scaffolds to promote healing in bone defects in pigs. These were all invaluable experiences that helped me develop as a scientist and prepare for further studies in animal science. Completing the project on comparing two embedding systems for follicle culture was the first project I was the lead on, and because of it, I learned a lot about structuring research, solving problems, and examining data, all of which I will need to use for my next endeavors as a graduate student.