The combined effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and the phytoestrogen genistein on steroid hormone secretion, AhR and ERß expression and the incidence of apoptosis in granulosa cells of medium porcine follicles.

Low doses of endocrine disrupting chemicals (EDCs) used in combination may act in a manner different from that of individual compounds. The objective of the study was to examine in vitro effects of low doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 100 pM) and genistein (500 nM) on: 1) progesterone (P4) and estradiol (E2) secretion (48 h); 2) dynamic changes in aryl hydrocarbon receptor (AhR) mRNA and protein expression (1, 3, 6, 24 and 48 h); 3) dynamic changes in estrogen receptor ß (ERß) mRNA and protein expression (1, 3, 6, 24 and 48 h); and 4) induction of apoptosis in porcine granulosa cells derived from medium follicles (3, 6 and 24 h). TCDD had no effect on P4 or E2 production, but potentiated the inhibitory effect of genistein on P4 production. In contrast to the individual treatments which did not produce any effects, TCDD and genistein administered together decreased ERß and AhR protein expression in granulosa cells. Moreover, the inhibitory effect of TCDD on AhR mRNA expression was abolished by genistein. The treatments did not induce apoptosis in the cells. In summary, combined effects of low concentrations of TCDD and genistein on follicular function of pigs differed from that of individual compounds. The results presented in the current paper clearly indicate that effects exerted by low doses of EDCs applied in combination must be taken into consideration when studying potential risk effects of EDCs on biological processes.

The Effects of Phytoestrogen Genistein on Steroidogenesis and Estrogen Receptor Expression in Porcine Granulosa Cells of Large Follicles.

Genistein is a biologically active isoflavone with estrogenic or antiestrogenic activity which can be found in a variety of soy products. Since in pigs' diet soy is the main source of protein, genistein may affect the reproductive/endocrine systems in these animals. Genistein has been shown to alter porcine ovarian and adrenal steroidogenesis but the mechanism of this action is still not clear. It is known that genistein binds to both estrogen receptor alpha (ERα) and estrogen receptor beta (ERß), although it has a higher affinity to ERß. Moreover, this phytoestrogen was demonstrated to posses the activity of protein tyrosine kinase (PTK) inhibitor. The aim of the study was to examine the in vitro effects of genistein on: (1) progesterone (P4) and estradiol (E2) secretion by porcine luteinized granulosa cells harvested from large follicles, and (2) the mRNA and protein expression of ERa and ERß in these cells. In addition, to verify the role of PTK-dependent mechanisms possibly involved in genistein biological action, we tested the effects of lavendustin C, the nonsteroidal PTK inhibitor, on granulosa cell steroidogenesis. Genistein significantly inhibited P4 and did not affect E2 secretion by porcine luteinized granulosa cells isolated from large follicles. Lavendustin C did not affect basal steroids secretion by examined cells. Genistein did not alter ERa but increased ERß mRNA levels in the cultured porcine granulosa cells. In contrast to medium follicles, the expression of ERß protein was unaffected by genistein in granulosa cells of large follicles. To conclude, the soy phytoestrogen genistein acts directly on the porcine ovary to decrease progesterone production and to increase the expression of ERß mRNA. Moreover, genistein-induced changes in follicular steroidogenesis and granulosal sensitivity to estrogens in pigs may depend on maturity of the follicles.

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and phytoestrogen genistein on the activity and the presence of steroidogenic enzyme proteins in cultured granulosa cells of pigs.

Environmental estrogens such as dioxins (e.g. 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD) and phytoestrogens (e.g. genistein; G) are known to influence endocrine and reproductive processes in humans and animals. Because living organisms are usually exposed to small, non toxic, doses of dioxins and phytoestrogens, the aims of the study were to determine the effects of small, environmentally relevant doses of TCDD (100pM) and/or genistein (500nM) on: (1) the activity of steroidogenic enzymes (cholesterol side-chain cleavage enzyme, P450scc; 3ß-hydroxysteroid dehydrogenase, 3ß-HSD and aromatase, P450arom) and (2) amount of protein of the enzymes in granulosa cells isolated from medium and large ovarian follicles of pigs. To determine the activity of the enzymes, the incubation medium was supplemented with specific steroid substrates (25-hydroxycholesterol; pregnenolone; testosterone) of particular steroidogenic enzymes (P450scc, 3ß-HSD and P450arom, respectively). Subsequently, the production of progesterone (P450scc and 3ß-HSD) or estradiol (P450arom) was compared in the presence and absence of the appropriate steroid precursor. Neither genistein nor genistein combined with TCDD affected activity of P450arom and relative amounts of steroidogenic enzyme proteins in the examined granulosa cells of pigs. In contrast, genistein alone and in combination with TCDD decreased P450scc and 3ß-HSD activity as well as progesterone production in granulosa cells isolated from medium and large follicles of pigs. Because TCDD alone did not affect steroid hormone production or enzyme activity, the above effects should be ascribed solely to genistein. It appears that the effects of the examined doses of TCDD and genistein on granulosal cell functions were not additive.

Biochanin A affects steroidogenesis and estrogen receptor-ß expression in porcine granulosa cells.

Biochanin A, similar to other isoflavones, is present in soy and soy-based food, but predominantly in red clover. Red clover extract and biochanin A were reported to affect reproductive processes as well as to demonstrate menopause relief and anticancerogenic properties. Because porcine granulosa cells provide a suitable in vitro model for studying the intracellular mechanism of phytoestrogen action in the ovary, the objective of the study was to evaluate the in vitro effects of biochanin A on the following: (1) progesterone (P4) and estradiol (E2) secretion by granulosa cells, (2) viability of the granulosa cells, and (3) mRNA and protein expression of estrogen receptors α (ERα) and ß (ERß) in the granulosa cells harvested from both medium (3-6 mm) and large (≥8 mm) porcine ovarian follicles. RIA, alamarBlue assay, reverse transcriptase-polymerase chain reaction, and immunocytochemistry were used in the study to address the objectives. Biochanin A significantly inhibited P4 and did not affect E2 secretion by porcine granulosa cells regardless of the size of follicles that served as the source of the cells. Cell viability was not affected by the treatment. Biochanin A did not alter ERα and ERß mRNA levels in the cultured porcine granulosa cells. In contrast, this isoflavone increased (P < 0.05) the immunoexpression of ERß in the cells from both follicle types. In summary, biochanin A, similar to genistein and daidzein, affects follicular steroidogenesis and ER expression. Its effect on ERß protein was more intense compared with other previously examined phytoestrogens.