Inhibition of Maturation of Domestic Cat Oocytes by In vitro Culture with Granulosa Cells
World Small Animal Veterinary Association World Congress Proceedings, 2003
Tuempong Wongtawan; Busaban Chaisalee
Faculty of Veterinary Science, Mahidol University (Salaya Campus)
Puthamonthon, Nakhonpratom, Thailand

Objective

The aim of the study was to investigate whether granulosa cells have an effect on maturation of domestic cat oocyte.

Material & Methods

Fresh granulosa cells were obtained by aspiration of follicles and incubated with 1% hyaluronidase for cell disaggregation for 15 minutes. Cells were counted by using Makler counting chamber (Sefi Medical Instrument., Haifa, Israel) and washed in culture medium 4 times prior to co-culture with oocyte in vitro. Oocytes was cultured with granulosa cells at 3 different concentrations (5x105, 1x106, 2x106 cells/ml) in 75 µl droplets of IVM medium which comprised Dulbecco's Modified Eagle Medium (DMEM; Gibco BRL., Grand Island, NY, U.S.A.) supplemented with 1 mM pyruvate, 1 mM lactate, 0.4% BSA, 1 µg/ml LH, 1 µg/ml FSH, 1 µg/ml Estradiol, 100 IU/ml penicillin, 100 IU/ml streptomycin (Sigma Co) and incubated in a 5%CO2 incubator at 39 °C with high humidity for 34-36 hours. After 34-36 hours, cumulus cells were removed by incubation with 1% hyaluronidase and mechanical aspiration. Oocytes were observed under stereomicroscope for assess meiosis stage and finally confirm by using Hoechst 33258 stain and observed with fluorescent microscope.

Results

The result showed that the maturation rate of oocytes coculture with granulosa cell in concentration 2x106 cells/ml was statistically lower (P<0.05) than control group. Most of immature oocytes arrested at GVBD and M I stage (59%).

Moreover the percentage of immature oocytes was significantly (P<0.05) increased during the 2x106 granulosa cells co-culture. No significant difference between control and treatment group in percentage of degeneration and parthenogenesis.

Conclusion

In the present study demonstrated that oocytes co-cultured with granulosa cells at the concentration of 2x106 cells/ml could inhibit the in vitro maturation in domestic cats while granulosa cell at the concentration of 5x105 and 1x106 cells/ml did not affect IVM. The inhibitory effect depended on the concentration of granulosa cells. The increase in number of granulosa cells resulted in inhibition of oocyte maturation in domestic cats.

Treatment

Metaphase II
(%)

GVBD
(%)

Degeneration
(%)

Parthenogenesis
(%)

DMEM(control)

62/101 (61)a

20/101 (20)ab

17/101 (17)

2/101 (2)

DMEM+Granulosa 5x105 cells/ml

53/90 (59)a

26/90 (29)b

10/90 (11)

1/90 (1)

1x106 cells/ml

68/114 (60)a

16/114 (14)a

28/114 (25)

2/144 (1)

2x106 cells/ml

32/96 (33)b

48/96 (50)c

12/96 (13)

4/96 (4)

a-c Values with different superscripts show significant difference among groups of treatment (column) (P<0.05)

Supported by Faculty of Veterinary Science, Mahidol University.

Speaker Information
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Tuempong Wongtawan
Faculty of Veterinary Science, Mahidol University (Salaya Campus)
Puthamonthon, Nakhonpratom, Thailand


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