Cyclic Change in 3α-Hydroxysteroid Dehydrogenase in Rat Ovary during the Estrous Cycle

Abstract

3 alpha-Hydroxysteroid dehydrogenase (3 alpha-HSD) activity and content in the rat ovary were measured at various stages of the estrous cycle, and the enzyme protein in the ovary was localized by immunohistochemistry. The cyclic change of ovarian 3 alpha-HSD activity towards 5 alpha-dihydrotestosterone (5 alpha-DHT) as a substrate was characterized by two peaks. The first peak occurred at 0800 h on proestrus; then the reductase activity decreased and reached its minimum at 2000 h on proestrus. Thereafter, it gradually increased, reaching the second peak (170% of the value at 2000 h on proestrus) at noon of estrus. Quantitative analysis by immunoblotting revealed that the alteration in 3 alpha-HSD content in the rat ovary during the estrous cycle was essentially similar to that in 5 alpha-DHT reductase activity. Changes in the reductase activities towards 5 alpha-androstane-3,17-dione and 5 alpha-DHT and in the dehydrogenase activity towards androsterone in the ovary were entirely different from those in the 5 alpha-DHT reductase activity and 3 alpha-HSD content; on the other hand, the change in carbonyl reductase activity towards p-nitroacetophenone was similar to changes in 5 alpha-DHT reductase activity and 3 alpha-HSD content. Therefore, p-nitroacetophenone may be a useful substrate, instead of 5 alpha-DHT, for detection of 3 alpha-HSD activity at a high sensitivity, since the p-nitroacetophenone reductase activity was 10-fold higher than the 5 alpha-DHT reductase activity. The enzyme was primarily localized in the granulosa cells and CL cells. At 2000 h on proestrus, however, the overall intensity of immunostaining in the granulosa cells of the Graafian follicles was markedly diminished. In addition, immunoreactivity in the CL cells at 0800 h on estrus was observed only in the cells outlining the CL in some cases.

Influence of mycotoxin zearalenone and its derivatives (alpha and beta zearalenol) on apoptosis and proliferation of cultured granulosa cells from equine ovaries

“…However, it should be noted a trend in growth increasing, evaluated as S+G 2 /M proliferation peak, that was affected by 1 × 10 -2 and 1 × 10 -3 μM of ZEA and α-ZOL, respectively. The observed variability could be related to the different levels of HSDs in GS cultured in each experiment, as reported by other authors [30]. In addition, seasonal (light, temperature), environmental (stress, diet) and hormonal factors (including the enzymatic contents) as well as the holding time and temperature can influence the variability observed during experiments carried out with equine GCs [31].…”

“Rapid actions of the neurosteroid allopregnanolone on ovarian and hypothalamic steroidogenesis: Central and peripheral modulation”

“…The enzymatic activity was measured spectrophotometrically. The assay measured the reduction of NAD+ or the oxidation rate of NADPH cofactor at 340 nm, respectively, as an increase in absorbance in 1 minute at 37°C . The values were expressed as mU mg protein ‐1 min ‐1 .…”

“…The assay measured the reduction of NAD+ or the oxidation rate of NADPH cofactor at 340 nm, respectively, as an increase in absorbance in 1 minute at 37°C. 61,62,64 The values were expressed as mU mg protein -1 min -1 . The method of Lowry et al 65 was used for protein determination with BSA as the standard solution.…”

A single dose of allopregnanolone affects rat ovarian morphology and steroidogenesis

“…The enzymatic activities were assayed spectrophotometrically using a Zeltec spectrophometer. The assay of each enzyme measured the reduction of NAD+ or the oxidation rate of NADPH at 340 nm respectively (Kawano et al 1988, Takahashi et al 1995, Escudero et al 2012 as an increase in absorbance in 1 min at 37°C. A fraction of the enzymatic solution was reserved for protein quantification.…”