A model for studying LCAT reaction: In vitro cholesterol esterification in pig ovarian follicular fluid

Abstract

Phosphatidylcholine acyltransferase (lecithin:cholesterol acyltransferase or LCAT; EC 2.3.1.43) activity was found to be present in pig ovarian follicular fluid (POFF), in addition to pig serum (PS). The cholesterol esterification rate in both POFF and PS is linear with incubation time up to 2 hr. The mean absolute rate of POFF‐cholesterol esterification was 8.1±0.4 nmoles per ml per hr approximately one‐fourth of that in PS. However, the fractional rate (percent of labeled cholesterol esterified per hr) of POFF‐cholesterol esterification was similar to that observed in PS. There was little variation of absolute rate of cholesterol esterification in the fluid obtained from different sizes of follicles. Fatty acid or triacylglycerol did not participate in the reaction of cholesterol esterification in POFF. No appreciable change in enzymatic activity was found from storing POFF at 4 C for periods of time up to 24 hr or at −70 C up to 2 months, but activity was lost thereafter. On the other hand, PS showed a much longer period of stability (5 days at 4 C and 9 months at −70 C). A discrepancy between the fatty acid composition of cholesteryl esters formed by the LCAT reaction and the fatty acid composition at the C‐2 position of phosphatidylcholine led us to propose a two‐step mechanism for the LCAT reaction. It is concluded that the LCAT of POFF, as well as that of plasma, is specific for individual fatty acids rather than for the fatty acid composition of phosphatidylcholine. The fatty acid concentration of lysophosphatidylcholine decreased during prolonged incubation times (6 to 21 hr) suggesting that the increased lysophosphatidylcholine formed as a product of the LCAT reaction may be reused as substrate for the LCAT reaction or for hydrolysis by lysophosphatidylcholine hydrolase.

Bovine oviductal fluid components and their potential role in sperm cholesterol efflux

“…The LCAT activity is substantially lower in bovine plasma than in the plasma of other mammalian species (Ha and Barter, 1982) and may also be characteristic of the serum-derived fluid in this species. The activity of this enzyme was not measured, but it has been reported in other serum-derived reproductive fluids such as porcine and human follicular fluid (Yao et al, 1978;Langlais and Roberts, 1985). Also, Chol esterase may be present in OF (Menezo, 1975), in which case the high proportion of UC in OF HDL could be explained by enzymatic hydrolysis of the esterified sterol.…”

“…Net transfer of unesterified Chol from peripheral cell membranes is apparently accomplished by esterification of the sterol by 1ecithin:Chol acyltransferase (LCAT) on the surface of HDL, with apoprotein A1 as a cofactor (Dobiasova, 1983;Phillips et al, 1987). HDL is present in bovine (Savion et al, 1982), porcine (Chang et al, 19761, and human follicular fluid (Simpson et al, 1980;Langlais et al, 1988), whereas LCAT activity has been reported in human and porcine follicular fluid (Langlais and Roberts, 1985;Yao et al, 1978). High levels of serum HDL and LCAT activity are found in human serum at the time of ovulation (Barclay et al, 1965;Weinstein et al, 1978;Shaefer et al, 1983), whereas the uterus of estrogen-dominated rabbits is characterized by a negative sterol gradient (Davis, 1982).…”

A molecular membrane model of sperm capacitation and the acrosome reaction of mammalian spermatozoa

“…Alteration of the c/p ratio is considered to be the main passive physiological process which modulates membrane fluidity in animal tissues [Shinitzky, 19781 and this mechanism may be a component of sperm capacitation since it has been suggested that the entry of calcium into and across the sperm plasma membrane is not an active transport process [Clegg, 19831. The depletion of plasma membrane cholesterol can be achieved by plasma constituents such as high-density lipoproteins, lecithin: cholesterol acyl transferase, apolipoprotein A-1, and albumin [Glomset, 1968;Stein and Stein, 1973;Quarfordt and Hilderman, 19701. The presence of these constituents in capacitating media such as serum, oviductal, and follicular fluids [Shalgi et al, 1973;Beier;1974, Yao et al, 1978Simpson et al, 1980;Albers et al, 19811 raises the interesting possibility that cholesterol exchange between the sperm plasma membrane and capacitation factors occurs by a mechanism similar to that observed between the red cell membrane and plasma proteins. The resulting decrease in the c/p ratio in the sperm plasma membrane could account for the changes in osmotic properties [Bedford, 19641, the increased membrane permeability to calcium [Summers et al, 19761, and the increased calcium influx observed in capacitated spermatozoa [Singh et al, 19781.…”