¸S. Arikan, R.G. Rodway Animal Reproduction Science 64 2000 149–160 151
cyclodextrin on LH, dibutyryl cAMP and cholesterol-stimulated luteal progesterone production.
2. Materials and methods
Unless otherwise indicated all chemicals used in the experiments were obtained from Sigma. All solvents used in the b-carotene analysis were of HPLC grade.
2.1. Preparation of cyclodextrin-encapsulated β-carotene Preparation of water soluble b-carotene was carried out using following procedures.
Dimethyl-b-cyclodextrin 500 mg, Sigma was dissolved in water 1 ml. Crystalline b- carotene 40 mg was added, the mixture was gassed with argon and sonicated for 2 h
with frequent cooling. The mixture was then stirred rapidly for 24 h at room temperature and finally sonicated for another 2 h. The free carotene separated from the encapsulated
one by filtering through 0.22 membrane filters. Concentration of b-carotene in complex was 2.7 mgg b-cyclodextrin. Cholesterol in cyclodextrin preparation was obtained from
Sigma.
2.2. β-Carotene analysis The quantitative determination of b-carotene in the cyclodextrin complex was performed
by HPLC using a C-18 reverse-phase column Resolve, Millipore and elution with a mo- bile phase consisting of 50:45:5 vvv methanol–acetonitrile–tetrahydrofuran Oliver and
Kafwembe, 1992.
2.3. Preparation and incubation of luteal cells Heifer ovaries judged to be in the mid-luteal phase Ireland et al., 1980 were collected
from the local abattoir immediately after slaughter and transported to the laboratory in ice-cold culture medium. Mixed luteal cells small and large were isolated by collagenase
digestion as described by O’Shaughnessy and Wathes 1985. Cell viability as determined by trypan blue exclusion was between 90 and 95. Luteal cells were stained for 3b-HSD as
described by Bao et al. 1995. Cells with a positive stain for 3b-HSD 1 × 10
5
cellswell were cultured in 5 CO
2
95 air at 37
◦
C in foetal bovine serum pre-treated six-well plastic culture dishes in 2 ml serum-free culture medium Dulbecco’s Modified Eagle’s and HAM’S
F-12, 1:1 vv with 15 mmoll HEPES containing penicillin 100 unitsml, streptomycin 100 mgml, fungizone 2.5 mgml and ITS premix 50 ngml insulin, 50 ngml transferrin
and 50 pgml selenium. Medium was replaced after 24 h, thereafter was changed every 48 h. Treatments were started on day 3 and maintained for either 5 or 11 days. Results presented
in each figure are means ±S.E.M. of three corpora lutea. Within each replicate, each treatment consisted of six separate cell wells. All wells contained equal concentrations of
cyclodextrin, either as a complex with b-carotene or cholesterol or as free cyclodextrin. Used medium was stored frozen at −20
◦
C until assayed for progesterone by radioimmunoassay.
152 ¸S. Arikan, R.G. Rodway Animal Reproduction Science 64 2000 149–160
The mean extraction efficiency was 94.2, the limit of sensitivity was 6.1 pgtube and the intra- and inter-assay coefficients of variation were 7.31 and 8.87, respectively.
2.4. Statistical analysis Different treatments were assessed by analysis of variance ANOVA and Duncan’s
multiple range test. Significance was defined as P 0.05. All statistical analysis were carried out using the Statistical Package for the Social Sciences SPSS. All results are
reported as means ± S.E.M.
3. Results
