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
3.1. Effect of cyclodextrin-encapsulated β-carotene on basal or cholesterol-stimulated progesterone production by luteal cells
The first experiment investigated the influence of cyclodextrin-encapsulated b-carotene alone 2 mmoll or in combination with cholesterol 25 mgml on progesterone production
by bovine luteal cells. At the start of treatment on day 3 of culture, as expected, there was no significant difference between the treatment groups in terms of progesterone production
on day 3 Fig. 1. On day 5 treatment of cells with either b-carotene alone, cholesterol alone or with cholesterol plus b-carotene resulted in significant stimulation P 0.01.
However, the combined treatment caused significantly more stimulation P 0.01 than the individual treatments. However, by day 7 cells treated with b-carotene alone or b-carotene
plus cholesterol were producing no more progesterone than the control. Treatment with
Fig. 1. Effect of cyclodextrin-encapsulated b-carotene on basal or cholesterol stimulated progesterone production by bovine luteal cells. Treatment was started on day 3 of culture. Control
h
, 2 mmoll b-carotene , 25 mgml cholesterol or 2 mmoll b-carotene plus 25 mgml cholesterol . Within each day, different letters indicate
significant differences P 0.01.
¸S. Arikan, R.G. Rodway Animal Reproduction Science 64 2000 149–160 153
Fig. 2. Effect of cyclodextrin-encapsulated b-carotene on basal progesterone secretion by bovine luteal cells. Control
h
, 0.1 mmoll , 1 mmoll b-carotene. Treatment was started on day 3. Within each day different letters indicate significant differences P 0.01; ND: not detectable.
cholesterol alone reversed the decline in progesterone production seen in the control and other experimental groups and resulted in progesterone production being approximately
10-fold greater than controls. The effects of incubation with two lower concentrations of b-carotene encapsulated in
cyclodextrin are shown in Fig. 2. As found previously, the production of progesterone in control incubations fell steadily with time and by day 11 was less than 20 of the day 3 value.
By day 5 treatment with 0.1 mmoll b-carotene had not affected progesterone production, however, the same concentration resulted in significant stimulation P 0.05 on days 7, 9
and 11. Addition of a higher concentration of b-carotene 1 mmoll resulted in a significant stimulation on day 5, but caused a rapid decline in progesterone production by day 7 and
on days 9 and 11 progesterone production was undetectable.
Fig. 3 illustrates the effects of 0.1 and 1 mmoll b-carotene in the presence of cyclodextrin- encapsulated cholesterol on progesterone production. Again all treatments, including choles-
terol, were started on day 3 of incubation. Treatment of cells with cholesterol 25 mgml alone resulted in a significant stimulation on all treatment days P 0.001. Combined
treatment with cholesterol plus b-carotene 0.1 mmoll produced significantly more stimu- lation than cholesterol alone. However, the higher concentration of b-carotene 1 mmoll in
combination with cholesterol resulted in significant P 0.001 inhibition of progesterone production which eventually fell to undetectable levels by day 9 of incubation.
3.2. Effect of cyclodextrin-encapsulated β-carotene on LH or dbcAMP stimulated progesterone production
The previous series of experiments had demonstrated that b-carotene with or without cholesterol both encapsulated in cyclodextrin would at low concentration stimulate luteal
progesterone production until at least day 11 of culture. The following two experiments
154 ¸S. Arikan, R.G. Rodway Animal Reproduction Science 64 2000 149–160
Fig. 3. Effects of cyclodextrin-encapsulated b-carotene on cholesterol-stimulated progesterone production by bovine luteal cells. After the initial incubation for 3 days without treatment, cells were then incubated for a further
8 days with the following treatments: control
h
, 25 mgml cholesterol , 0.1 mmoll b-carotene plus with 25 mg ml
− 1
cholesterol or 1 mmoll b-carotene plus with 25 mgml cholesterol . Within each day, different letters indicate significant differences P 0.05.
investigated the effects of b-carotene when used in combination with either LH or dibutyryl cAMP to maintain steroidogenesis. Again treatment was started on day 3. As shown in
Fig. 4 both dbcAMP 1 mmoll and LH 100 ngml as expected significantly stimulated progesterone production throughout the incubation period P 0.01. Unfortunately, due to
Fig. 4. Effect of cyclodextrin-encapsulated b-carotene on LH and dbcAMP stimulated progesterone production by bovine luteal cells. Cells were incubated in medium without treatment for the initial 3 days. On day 3 the following
treatments were applied: 100 ngml LH
h
, 100 ngml LH plus 25 mgml cholesterol plus , 100 ngml LH plus 25 mgml cholesterol plus 0.1 mmoll b-carotene or 1 mM dbcAMP , 1 mM dbcAMP plus 25 mgml
cholesterol plus , 1 mM dbcAMP plus 25 mgml cholesterol plus 0.1 mmoll b-carotene .Within each day, different letters indicate significant differences P 0.01.
¸S. Arikan, R.G. Rodway Animal Reproduction Science 64 2000 149–160 155
Fig. 5. Effect of cyclodextrin-encapsulated b-carotene on LH stimulated progesterone production by cultured luteal cells. Treatment was started on day 3, on which cells were cultured with medium under basal conditions
h
, with 0.1 mmoll b-carotene , 100 ngml LH or with 0.1 mmoll b-carotene plus 100 ngml LH . Every groups including basal contained 25 mgml cholesterol. Within each day, groups with different letters are
significantly different P 0.05.
limitations on the number of cells available, no dose-response data for LH or dbcAMP could be obtained. However, treatment of cells with a combination of cholesterol and either LH or
dbcAMP resulted in a more than three-fold increase in progesterone production compared with LH or dbcAMP alone. Surprisingly, cells treated with b-carotene 0.1 mmoll plus LH
or dbcAMP produced significantly P 0.001 less progesterone from day 7 until the end of culture period than did those treated with LH or dbcAMP alone.
The final experiment was performed to confirm the previous observation that b-carotene when given alone plus cholesterol caused a stimulation of progesterone production, but
when given in combination with LH plus cholesterol its effect was inhibitory. Fig. 5 indicates that b-carotene 0.1 mmoll plus cholesterol caused an increase in progesterone
production which was significant from day 9 onwards, while when given together with LH it substantially inhibited the stimulation given by LH alone.
4. Discussion
