- Kathryn Polkoff
- A comparison of two embedding systems for follicle culture
- Animal Sciences
Describe
your research
experience
My research interests include a wide variety of disciplines in Animal Science with a base in reproductive biology, including reproductive techniques, regenerative medicine, and transgenic engineering.
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 pre-antral 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 12-117F) 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 39°C 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.
| |
Control
|
Alginate 0.5%
|
Alginate 1%
|
Agar 1%
|
Agar 2%
|
|
Day 2
|
23.4 ( 38.1)
|
31.4 (43.1)
|
42.4 (36.8)
|
3.8 (118.8)
|
29.9 (45.4)
|
|
Day 4
|
11.2 (39.5) a
|
35.7 (44.7)
|
77.9 (17.6) b
|
40.0 (89.3)
|
76.2 (48.6) b
|
a,b Least square means (± SD) within each row without common superscripts differ (P< 0.05).
As one of the recipient's of OUR's Summer Undergraduate Research Fellowships (2015), the results of Kathryn's research can be found here.
