Ž .
LDL also supply other fat soluble substances such as cholesterol, vitamin A and Ž
. vitamin E to the corpus luteum Ribaya-Mercado et al., 1993; Aten et al., 1994 .
However, several previous studies have used water miscible solvents such as THF Ž
. Ž
. Bertram et al., 1991 and DMSO Young et al., 1995 to supply b-carotene to cultured
luteal cells. The aim of the present study was to compare the use of THF and DMSO with that of HDL as a mean of supplying b-carotene to luteal cells, and to examine the
interaction between b-carotene and LH and dbcAMP on progesterone production.
2. Materials and methods
2.1. Preparation of lipoproteins A 10-day-old Holstein bull calf was fed twice daily with milk replacer containing 2.5
Ž .
g of b-carotene Roche, UK per feeding for 10 days. Blood was collected to obtain HDL after killing the animal. Serum was separated by centrifugation. HDL was isolated
from serum between densities of 1.061 and 1.215 by sequential ultracentrifugation Ž
. Havel et al., 1955 using potassium bromide and sodium chloride for density adjust-
ment. Centrifugation was accomplished using a Beckman 70Ti fixed angle rotor for 48 h Ž
. at 52,000 rpm. 200,000 gav and 158C in a Beckman Model LS-55 ultracentrifuge.
HDL containing low b-carotene was prepared similarly from serum collected from an unsupplemented calf. Cholesterol in the lipoprotein fractions was determined using the
Ž cholesterol-oxidase: peroxidase method. The assay was performed using a kit Roche
. Diagnostic, NJ
based on the oxidation of cholesterol to cholest-4-ene-3-one and hydrogen peroxide and the subsequent conversion of 4-aminoantipyrine to the dye
quinoneimine. The absorbance was measured at 500 nm. HDL obtained from Sigma was also used for quality control. The b-carotene concentrations in HDL isolated from serum
containing high or low b-carotene were 12.4 and 0.44 mgrmg cholesterol, respectively. Lipoprotein fractions were sterilised by passage through a 0.22-mm millipore filter and
stored at y408C until use.
2.2. Isolation and culture of boÕine luteal cells Bovine ovaries were collected immediately after slaughter from the local abattoir and
transported to the laboratory in ice-cold phosphate-buffered saline within 30 min. Ovaries were normally from non-pregnant Friesian Holstein cattle judged to be at
Ž .
mid-cycle by the criteria of Ireland et al. 1980 . All chemicals were obtained from Ž
. Sigma. Mixed luteal cells small and large were isolated from mid-luteal heifer ovaries
Ž .
by collagenase digestion as described by O’Shaughnessy and Wathes 1985 . Luteal Ž
. cells were stained for 3b-hydroxysteroid dehydrogenase 3b-HSD as described by Bao
Ž .
et al. 1995 . Cells were counted using a hemocytometer and viability was estimated Ž
5 5
. using trypan blue exclusion. Cells 1 = 10 y 2 = 10
with a positive stain for 3b-HSD Ž
were cultured in foetal bovine serum pre-treated six-well plastic culture dishes 35 mm, .
Ž Sterillin, Feltham, England with 2 ml or 4 ml in b-carotene uptake and depletion
. Ž
experiments serum-free culture medium Dulbecco’s Modified Eagle’s and HAM’S Ž
. Ž
. F-12,1:1 vrv with 15 mmolr1 HEPES containing penicillin 100 unitsrml , strepto-
Ž .
Ž .
Ž mycin 100 mgrml , fungizone 2.5 mgrml and ITS premix 50 ngrml insulin, 50
. ngrml transferrin and 50 pgrml selenium in a humidified incubator that contained 95
O and 5 CO . Medium was replaced after 24 h. Unless otherwise indicated, thereafter
2 2
medium was changed every 48 h. Each treatment consisted of six separate cell wells. The protocol for each experiment is described in Section 3. Cells were incubated for a
maximum of 11 days. Used medium was stored frozen at y208C until assayed for progesterone by radioimmunoassay. The mean extraction efficiency was 94.8, the limit
of sensitivity was 6.28 pgrtube and the intra- and inter-assay coefficients of variation were 7.48 and 8.73, respectively.
2.3. b-Carotene analysis 2.3.1. Extraction of b-carotene from HDL, luteal tissue and cultured cells
Ž To extract b-carotene from HDL, 2 ml ethanol containing 0.01 butylated hydroxy-
Ž .
. toluene BHT as an anti-oxidant was added to 100 or 250 ml HDL to precipitate
protein. The mixture was vortexed and extracted three times with 3 ml of hexane. The combined supernatants were evaporated under a stream of N
and the residue was
2
redissolved in ethanol. Ž
. Cells were removed from the culture dishes by trypsinization with 0.05 wrv
Ž .
trypsin in phosphate-buffered saline containing 0.02 wrv EDTA. Extraction of Ž
. Ž
6
. b-carotene from luteal tissue 500 mg and incubated luteal cells 10
cells was Ž
. performed as described by Schmitz et al. 1993 .
2.4. Chromatography All solvents used were of HPLC grade. The HPLC system used for b-carotene
analysis consisted of a Spectra-physic SP8700 solvent delivery system and an SP8750 Ž
. Ž
. manual injector Spectra-physic, San Jose, CA, USA . Samples 50 ml were injected
Ž onto a 10-mm, spherical, C-18 reverse-phase column
30 cm = 3.9 mm; Resolve, .
Ž .
Millipore and eluted with a mobile phase consisting of 50:45:5
volrvolrvol Ž
. methanol–acetonitrile–tetrahydrofuran Oliver and Kafwembe, 1992 , with a flow rate
of 2 mlrmin. b-Carotene was detected at wavelength of 452 with a Spectraflow 757 Ž
. model variable wavelength detector Kratos Analytical Instruments, USA . Peak areas
were compared to those of authentic standards for quantification. 2.5. Statistical analysis
Ž .
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 was
Ž .
carried out using the Statistical Package for the Social Sciences SPSS . All results are reported as means SEM.
3. Results
