COLOCACION DE ELECTRODOS

Cholesterol is converted to progesterone via the pathway shown in figure 4.2.6.

2 0 a hydroxycholesterol

cholesterol --- 22R hydroxycholesterol — ^ pregnenolone — ^ progesterone 20a,2 2 R dihydroxy cholesterol

P450CSCC 3 P- H S D

Figure 4.2.6. Pathway o f P formation from cholesterol.

It was initially intended that both the conversion of cholesterol to pregnenolone and pregnenolone to P would be studied. This proved impossible because the amount of P formed from cholesterol, 25-OHcholesterol and LDL in theses studies was insufficient for accurate measurement of P450cscc. The reason for the low levels of P production on the addition of 25-OHcholesterol even when the number of cells was raised to 1,000,000 viable cells per well and the concentration of 25-OHcholesterol was as high as 80 jimol/l is uncertain. The levels of P produced by cells incubated in DMEM alone were not significantly different to those incubated with cholesterol or 25-OHcholesterol. These findings are in accord with another study which also found that cholesterol was a poor precursor for P production in human syncytiotrophoblast cell cultures (Paul, Das, Jailkhani and Talwar 1987). A possible explanation is that cholesterol or 25- OHcholesterol is not reaching the enzyme system either because its transfer across the cell membrane or its transport within the cell to the enzyme system within the mitochondria has been in some way affected. In the case of 25-OHcholesterol it is unlikely that it is the transfer across the cell membrane that is affected as this derivative of cholesterol has been used by other researchers because of its greater solubility than

cholesterol in aqueous medium and has been shown to be an effective steroidogenic substrate in dispersed rat luteal cells (Sinensky 1981; Toaff, Schleyer, and Strauss 1982). Indeed a study by Tuckey (1992) using isolated human placental mitchondria to measure P-450cscc activity has shown that 25-OHcholesterol is a poor substrate for pregnenolone synthesis in this cellular organelle. The other possibility is that the P- 450CSCC enzyme system is not functioning properly in isolated cells. However, it seems unlikely that the enzyme system was damaged by the isolation process as both the aromatase sytem and 3p-HSD sytem are functioning normally. The cytotrophoblast P- 450cscc may not be "switched on'" or it may be present within the isolated cells but only at very low levels. Levels of P-450cscc have been reported to be lower in cultured cytotrophoblast cells than in placental homogenates suggesting that the synthesis of this enzyme could well be induced as syncytiotrophoblasts form (Tuckey, Kostadinovic and Cameron 1994). Kliman and colleagues (Kliman, Nestler, Sermasi, Sanyer and Strauss 1986) were able to show an increase in P levels produced by purified cytotrophoblast cells by 2-8 fold in four hours with the addition of 50 fimol/l 25-OHcholesterol. Nestler and Williams (1987) incubated cytotrophoblast cells with 50 |Limol/l 25-OH cholesterol and also obtained measurable levels of P (approximately 52 pmoles/million cells/24 hours). In both cases much less than 1% of the substrate was converted to P in 24 hours. The values obtained by both sets of reseachers are very low considering the concentration of precursor used. The discrepancies between this study and those by Kliman et al (1986) and Nestler and Williams (1987) are difficult to explain. It is of course possible, though unlikely that the RIA assay employed in this study was not as sensitive as that used by Kliman et al (1986) and Nestler and Williams (1987) or it could be that there was an appreciable amount of cross-reactivity with other substances which resulted in the "measurable" levels obtained with the assays of other researchers.

The fact that LDL and 25-OHcholesterol proved to be poor precursors for P production in this cell system poses the possibility that the major precursor for P may well not be LDL but could be cholesterol sulphate, a substrate that may be readily available to placental cells in vivo. Indeed Tuckey et al (1994) found that cholesterol sulphate was a

better precursor for P production by cytotrophoblast cells and mitochondria than LDL or other intermediates in cholesterol side chain cleavage.

Because of the low level of conversion of cholesterol to pregnenolone it was not possible to measure P450cscc activity in these studies. The addition of pregnenolone did result in P production showing that the 3p-HSD enzyme was functioning within these cells. The studies by Mason et al (1993) indicated that the levels of mRNA for 3p- HSD in cytotrophoblasts were undectable after isolation of these cells but increased with time in culture. However their findings do not completely accord with preliminary experiments in this study in which substantial progesterone formation was detectable in the media from cells incubated for only 2 hours with pregnenolone (mean levels from

two placentae incubated in triplicate wells were 560 fm oles/100,000 viable cells). However this does not exclude the possibilty that synthesis or activation of 3P-HSD is occurring with time in culture. In this study cytotrophoblast cells were isolated from three placenta and cultured for up 96 hours. Cytotrophoblast cells from each of the three placentae were incubated immediately after purification with pregnenolone for 24 hours and then frozen. Other cell preparations from the three placentae were incubated with DMEM alone for 72 hours before the addition of pregnenolone to the culture medium. The cells were then incubated with the precursors for a further 24 hours. The levels of P produced by those cells incubated in DMEM alone for 72 hours before the addition of pregnenolone were higher (means ranged from 2.71 to 10.47 nmol/100,000 viable cells/24 hour) than those produced by cells incubated with this precursor immediately after purification (means ranged from 1.12-8.65 nmol/100,000 viable cells/24 hour), suggesting that the activity or the amount of enzyme is increasing in time in culture. In the cell preparations obtained from one of the placentae studied the enzyme system was not saturated even at pregnenolone concentrations of 80 pmol/l. However even at concentrations as low as 5 nmol only around 10% of the precursor was being transformed to P with the percentage dropping to about 3% at concentrations of 10 nmol/1 which is in agreement with the results of Nestler (Nestler and Williams 1987) and suggests that it is the entry of pregnenolone into the cell or its transport within the

cell that is the rate limiting factor in this cell system. On incubating cytotrophoblast cells at a concentration of 6 |imol/l pregnenolone Nestler obtained mean levels of 649

pmol/million cells. In the present study mean levels of P produced by isolated cytotrophoblast incubated with 1 |imol/l pregnenolone were 330-440 pmol/million viable cells/24hours and those incubated with 10 |xmol/l pregnenolone were in the range 1600-3110 pmol /million viable cells/24 hours (results given in table 3.11).