Granulosa cell aromatase enzyme activity: Effects of follicular fluid from patients with polycystic ovary syndrome, using aromatase conversion and [¹¹C]vorozole-binding assays

Abstract

The local regulation of ovarian aromatase enzyme in polycystic ovary syndrome (PCOS) was studied with aromatase conversion and [11C]vorozole-binding assays to analyze aromatase activity, substrate-enzyme affinity and number of aromatase binding sites in non-cultured human granulosa cells (GC) incubated with different sources and preparations of follicular fluid (FF). Incubation with FF from women stimulated in in vitro fertilization cycles with follicle-stimulating hormone yielded higher conversion activity than with FF from healthy women and PCOS patients, paralleled with higher substrate affinity (lower Kd) than with FF from healthy women. In PCOS women, charcoal-pretreated FF yielded higher conversion, whereas the ether-pretreated FF yielded lower conversion activity, than with untreated PCOS FF. Both preparations of FF yielded higher affinity to substrate (lower Kd values) and the ether-pretreated FF a lower number of binding sites (Bmax). It seems that steroids with the presence of proteins in PCOS FF reduced aromatase conversion activity through decreased substrate affinity, whereas FF preparations devoid of proteins reduced the aromatase conversion activity mainly through blocking of aromatase active sites. Identification of specific agents responsible for this rapid regulation of aromatase function might help to understand normal regulation of the menstrual cycle and supposed imbalances of inhibitors/activators in PCOS.

Steroid profiles in ovarian follicular fluid in women with and without polycystic ovary syndrome, analyzed by liquid chromatography-tandem mass spectrometry

“…Thirteen steroids were quantitated, requiring only 40 mL of FF. The concentrations of total and individual androgens and Es, product-to-precursor ratios, and ROC curve plottings all indicated a higher activity of CYP17-linked enzymes increasing FF androgen levels combined with lower ovarian CYP19-linked enzyme (aromatase) activity lowering FF Es in women with PCOS, confirming previous data (5,11,17,18). The ratio (total androgens/5P), somewhat reflecting the C17-20 side chain cleaving enzyme activity, was significantly higher in FF from PCOS.…”

“…Furthermore, increased CYP17 and reduced CYP19-linked enzyme activity are in line with previous findings for CYP17 (17,18) and for CYP19 (11) activities in women with PCOS. In women diagnosed with PCOS, BMI was negatively associated with concentrations of total Es, E 2 , E 2 /T, and 17-OHP.…”

“…These findings agree with our report on GC aromatase enzyme activity after exposure to FF from women with PCOS, showing reduced GC aromatase conversion activity (E biosynthesis), partly by blocking the active site of the enzyme and partly by decreased substrate affinity (11). The lack of differences (although numerically lower) in FF E levels in two previous studies (19,20) might be explained by the low number of patients with PCOS (five) or the RIA methods used.…”

“…The study subjects were recruited by advertising in local media and investigated at the Donetsk Regional Center of Mother and Child Care, Donetsk, Ukraine, as described in detail elsewhere (11). Available FF from 27 women with PCOS and 21 regularly cycling women without PCOS were included.…”

“…Furthermore, restricting analysis to women with PCOS having more severe oligomenorrhea (<6 cycles/year), revealed similar differences and significance levels, as shown in Table 2. The PCOS and regularly menstruating women in this study were subjects from our previous study on ovarian aromatase enzyme regulation (11). Women in the regularly menstruating group were also included in our recent report, illustrating the methodology and feasibility of quantitating multiple steroids in FF using LC-MS/MS methods (10).…”