Insulin stimulates progesterone secretion to a greater extent than LH in early pregnant buffalo luteal cells cultured in vitro

In the present study, effect of insulin alone or in combination with LH on modulation of progesterone production by early pregnant buffalo luteal cells was reported. Luteal cells were isolated using collagenase and subsequently cultured in Ham'F-12 at 37 degrees C in an atmosphere of 5% CO2 and 95% humidified air. Small luteal cells (SLC, 12-23 mu) appeared as spindle shaped with eccentrically placed irregular nucleus, however, large luteal cells (LLC, 25-55 mu) were polyhedral or spherical in shape with centrally placed large round nucleus having one or two nucleoli. There was an abundance of cytoplasmic lipid droplets and a greater cytoplasmic to nuclear ratio as compared to SLC. Both small and large luteal cells were positive to 3 beta-HSD, a marker for steroidogenic capacity. Luteal cells attached to surface within 24 h of culture and appeared typical of epithelial cells with numerous cytoplasmic lipid droplets within the cytoplasm. These cells maintained the morphological characteristics throughout the culture period. Luteal cells were treated with insulin (0.05 IU/ml) and LH (10 ng/ml) alone or in combination for 7 days to study the effect on progesterone production. Morphology of luteal cells did not change with the addition of LH and insulin. Addition of insulin enhanced (P < 0.01) basal as well as LH stimulated progesterone production and also minimized loss of cell number by maintaining greater cell populations throughout the culture period as compared to control and LH treatment. In the absence of tropic stimulation, progesterone secretion decreased rapidly in the control group while addition of insulin greatly decreased the rate of decline. The findings of the present study reveal insulin enhances progesterone secretion by the luteal cells indicating its possible role to modulate corpus luteum function in buffalo. (C) 2013 Elsevier B.V. All rights reserved.