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The aim of this study was to examine the expression and regulation of type 1 17 beta-hydroxysteroid dehydrogenase (type 1 17-HSD) enzyme protein and mRNA, and 17-HSD activity in human granulosa cells. The cells were obtained from patients taking part in an in vitro fertilization programme. The cells from each patient were divided into two groups: cells obtained from preovulatory follicles (LGC = granulosa cells from large follicles > or = 18 mm in diameter), and cells from other visible follicles (SGC = granulosa cells from small follicles, less than 15 mm in diameter). The identity of 17-HSD enzyme protein expressed in human granulosa cells with placental cytosolic 17-HSD (type 1 17-HSD) was assessed by immunoblot analysis using polyclonal antibodies, and the enzyme was immunolocalized in the cytoplasm of granulosa cells. Type 1 17-HSD protein concentration, 17-HSD and cytochrome P450 aromatase (P450arom) activities and oestradiol (OE2) production in cells from LGC were significantly lower than the corresponding values obtained in SGC in the same patient (paired t-test). The type 1 17-HSD protein concentration, 17-HSD activity and P450arom activity were 140 +/- 16% (mean +/- S.E.M.), 121 +/- 22% and 113 +/- 26% higher in cells from SGC, which was also reflected in a 70 +/- 12% higher OE2 production in these cells. In freshly isolated cells from LGC or SGC, a high correlation between 17-HSD and P450arom activities was observed (r = 0.93, P < 0.001). In long-term cultured cells, type 1 17-HSD was stably expressed at least until day 9, while P450arom expression decreased. In addition, treatments with gonadotrophins did not affect type 1 17-HSD protein concentration and 17-HSD activity. In contrast to this, both P450arom activity and OE2 production were significantly increased (P < 0.05). The data, therefore, suggest that type 1 17-HSD and P450arom are expressed in parallel during the latest stages of follicular maturation but, in cultured granulosa-luteal cells, the enzymes are regulated by distinct mechanisms.

Antibodies against human placental 17 beta-hydroxysteroid dehydrogenase (17-HSD) and 17-HSD cDNA were used to study the expression of the corresponding enzyme in the immature rat ovary during follicular development and luteinization, which were induced by treating the animals with pregnant mare serum gonadotrophin (PMSG) or with PMSG followed by human chorionic gonadotrophin (hCG). Immuno-blot analysis indicated that the M(r) of the 17-HSD expressed in rat granulosa cells was 35,000, as previously shown for the human placental enzyme. In immunohistochemical studies of untreated immature rat ovaries, only the granulosa cells from small antral follicles were stained. One day after PMSG treatment, strong expression of 17-HSD was observed in the granulosa cells of growing Graafian follicles. A marked decrease in enzyme expression was observed in preovulatory follicles on day 2 of PMSG treatment, starting from the basal layers of granulosa cells and progressing toward the luminal cells. No 17-HSD expression was detected in luteinized follicles or corpora lutea 22 h after hCG injection. The stroma and theca cells were negative for 17-HSD staining. In Northern hybridization analyses, two 17-HSD mRNAs were detected (1.4 and 1.7 kb). The strongest expression for both mRNAs was detected after 1 day of PMSG treatment, coinciding with maximal immunostaining of the enzyme protein. Down-regulation of 17-HSD observed by immunohistochemistry was reflected in a similar decrease in mRNA expression and the signals were almost undetectable 22 h after hCG injection. Our data suggest that 17-HSD expression in rat granulosa cells is up-regulated during follicular development and, thereafter, the enzyme expression is down-regulated during luteinization.