In vitro growth of oocytes from primordial follicles isolated from frozen-thawed lamb ovaries.
A study was conducted to develop an in vitro culture system for growing sheep oocytes from isolated primordial follicles. Enzymatically isolated neonatal sheep primordial follicles were cultured in Waymouth MB752/1 medium containing BSA (3 mg/ml) + ITS (1%, v/v) over 28 days. In Experiment 1, primordial follicles (average diameter 40.2+/-0.60 microm) were cultured at densities of 20, 50 and 100 follicles per well. Less than 20% of the oocytes survived to day 28 but there was a significant (P < 0.05) increase in median oocyte diameter from day 2 to day 28 for oocytes cultured at the higher densities of 50 and 100 follicles. In Experiment 2, two methods to improve oocyte:granulosa cell associations were tested. Altering the fibronectin coating regime did not improve oocyte survival and growth. In contrast lectin-aggregated primordial follicles cultured on non-coated wells showed significantly (P < 0.05) improved oocyte survival to 50% and increased median oocyte diameter compared to non-aggregated follicles. In Experiment 3, the effect of KIT ligand (KL) at 0 ng/ml, 10 ng/ml and 100 ng/ml, on lectin-aggregated primordial follicles cultured on non-coated wells was tested. KL at 100 ng/ml significantly (P < 0.05) increased median oocyte diameter compared to non-treated controls but had no effect on oocyte survival. In addition, follicles cultured with 100 ng/ml KL expressed mRNA for AMH, a gene expressed only in granulosa cells of growing follicles. In conclusion, culture of lectin-aggregated primordial follicles supported the long-term survival and growth of oocytes from isolated sheep primordial follicles. Culture of lectin-aggregates with 100 ng/ml KL further increased oocyte growth and induced granulosa cell differentiation.
Muruvi
Wanzirai
W
School of Biology, University of Leeds, Clarendon Way, Leeds LS2 9JT, UK. wm64@cornell.edu
Picton
H M
HM
Rodway
R G
RG
Joyce
I M
IM
D000818:Animals; D000831:Animals, Newborn; D015925:Cryopreservation / Q000662:veterinary; D003470:Culture Media; D005260:Female; D006107:Granulosa Cells / Q000502:physiology; D007150:Immunohistochemistry / Q000662:veterinary; D037102:Lectins; D009865:Oocytes / Q000254:growth & development* / Q000502:physiology; D009924:Organ Culture Techniques / Q000379:methods / Q000662:veterinary*; D006080:Ovarian Follicle / Q000166:cytology* / Q000196:embryology; D010053:Ovary / Q000166:cytology / Q000502:physiology; D012756:Sheep
D003470:Culture Media; D037102:Lectins