Animal Reproduction Science 64 2000 149–160
Effect of cyclodextrin-encapsulated b-carotene on progesterone production by bovine luteal cells
¸S. Arikan
∗
, R.G. Rodway
Department of Animal Physiology and Nutrition, University of Leeds, Leeds LS2 9JT, UK Received 16 March 2000; received in revised form 2 August 2000; accepted 22 August 2000
Abstract
Experiments were conducted to examine the effect of cyclodextrin-encapsulated b-carotene
on basal or cholesterol cyclodextrin-encapsulated, LH and dibutyryl cyclic AMP dbcAMP- stimulated progesterone production by bovine corpus luteum cells isolated from mid-luteal heifer
ovaries by collagenase digestion. Cells were cultured with serum-free DMEMHam’s F12 medium in serum pre-treated plastic culture dishes for periods of up to 11 days. Medium was replaced
after 24 h and thereafter every 48 h.
b-Carotene was added to cultures in a carrier molecule, dimethyl-
b-cyclodextrin, to facilitate dissolution. All treatments were started on day 3 of cul- ture. Treatment of cells with 1 or 2
mmoll b-carotene resulted in sharp inhibition of progesterone production. On the contrary, treatment of cells with 0.1
mmoll b-carotene resulted in significant stimulation P 0.05 of both basal and cholesterol-stimulated progesterone secretion. The ef-
fect of b-carotene on LH or dbcAMP-stimulated progesterone production was also examined.
Treatment of cells with LH or dbcAMP always resulted in stimulation of progesterone secretion P 0.001. However, cells treated with LH plus
b-carotene or dbcAMP plus b-carotene both produced significantly P 0.01 less progesterone relative to those cells treated with LH or
dbcAMP alone on days 7, 9 and 11 of culture. These results indicate that b-carotene can enhance
luteal steroidogenesis when present at low concentrations but is inhibitory at higher concentrations and that encapsulation of
b-carotene in cyclodextrin is an effective method of supplying it to cells in culture. © 2000 Elsevier Science B.V. All rights reserved.
Keywords: b-Carotene; Cattle-endocrinology; Luteal cells; Cyclodextrin; Progesterone
1. Introduction
b -Carotene is present in extremely high concentrations in the bovine corpus luteum
CL O’Fallon and Chew, 1984; Holt et al., 1995, giving the CL its characteristic bright
∗
Corresponding author. Present address: Faculty of Veterinary Medicine, University of Kirikkale, Kirikkale 71100, Turkey. Tel.: +90-318-3573301; fax: +90-318-3573304.
E-mail address: sevket95hotmail.com ¸S. Arikan. 0378-432000 – see front matter © 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 4 3 2 0 0 0 0 0 2 0 2 - 5
150 ¸S. Arikan, R.G. Rodway Animal Reproduction Science 64 2000 149–160
yellow colour. As well as acting as a precursor for vitamin A there is increasing evidence that b-carotene may be necessary for optimal steroid production, possibly acting as an
anti-oxidant Young et al., 1995. It has been reported previously that b-carotene may effect luteal cell steroid production in vitro Pethes et al., 1985; O’Shaughnessy and Wathes, 1988
and in vivo Dembinski and Bronicki, 1994. Also, Graves-Hoagland et al. 1988 found that a positive relationship existed between in vitro bovine luteal cell progesterone production
and plasma b-carotene during the winter when plasma b-carotene concentrations are low in dairy herds. They showed that during the summer when plasma b-carotene is increased, this
relationship is lost. However, some studies in dairy cattle have failed to observe an effect of b
-carotene supplementation on plasma steroid hormone levels Folman et al., 1979; Wang et al., 1982.
Previous work in this laboratory has demonstrated that b-carotene will stimulate pro- gesterone production in bovine luteal cells maintained in tissue culture and also that the
b -carotene concentration in the culture medium becomes depleted during incubation and
that this occurs more rapidly when the cells are stimulated with either LH or cAMP Arikan and Rodway, 1997, 1998, suggesting that b-carotene is metabolised during the process of
steroidogenesis. As b-carotene is almost completely insoluble in water and most other polar solvents,
several methods have previously been used to deliver b-carotene to cells in culture. These include the use of water miscible organic solvents such as ethanol Gross et al., 1997,
dimethylsulphoxide Young et al., 1995 and tetrahydrofuran Bertram et al., 1991, Cooney et al., 1993. However, the use of such organic solvents may cause cytotoxicity, as well
as having effects on cell metabolism, proliferation and membrane permeability Yu and Quinn, 1994 and solvents such as DMSO have been demonstrated to stimulate progesterone
synthesis in incubated luteal cells Arikan and Rodway, 1997. b-Carotene is normally transported to the ovary incorporated in the lipid component of high density lipoprotein
HDL in the bovine O’Shaughnessy and Wathes, 1988. However, HDL also supplies other fat soluble substances such as cholesterol, vitamin A and vitamin E to the corpus
luteum Ribaya-Mercado et al., 1993; Aten et al., 1994. Although HDL has been used previously as a method of delivering b-carotene to luteal cells O’Shaughnessy and Wathes,
1988; Arikan and Rodway, 1997, the presence of these other substances in the lipoprotein preparations Ribaya-Mercado et al., 1993 and the variable content of b-carotene in the
control preparations leads to difficulties in the interpretation of the results. In view of these problems it was decided to investigate the use of cyclodextrins for the delivery of b-carotene
to luteal cells in vitro.
b -Cyclodextrins are cage-like molecules consisting of seven glucose units and have the
appropriate size to form inclusion complexes with many hormones, vitamins and drugs having a size compatible with the dimensions of the cavity Pitha, 1981; Uekama and Irie,
1987; Albers and Muller, 1992. Hydrophobic guest molecules such as b-carotene can be incorporated into the cavity of the cyclodextrin by displacing water. The resulting complex
is water-soluble, although the guest molecule can be released relatively easily and taken up by cells in culture.
The first objective of this study was to determine the effectiveness of cyclodextrin as an agent for the delivery of b-carotene to bovine luteal cells in tissue culture. The
second objective was to examine the effects of b-carotene encapsulated within
¸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
