AVPV neurons containing estrogen receptor-beta in adult male rats are influenced by soy isoflavones

Abstract

Background: Isoflavones, the most abundant phytoestrogens in soy foods, are structurally similar to 17beta-estradiol. It is known that 17beta-estradiol induces apoptosis in anteroventral periventricular nucleus (AVPV) in rat brain. Also, there is evidence that consumption of soy isoflavones reduces the volume of AVPV in male rats. Therefore, in this study, we examined the influence of dietary soy isoflavones on apoptosis in AVPV of 150 day-old male rats fed either a soy isoflavone-free diet (Phyto-free) or a soy isoflavone-rich diet (Phyto-600).

Results: The occurrence of apoptosis in AVPV was examined by TUNEL staining. The incidence of apoptosis was about 10 times higher in the Phyto-600 group (33.1 +/- 1.7%) than in the Phyto-free group (3.6 +/- 1.0%). Furthermore, these apoptotic cells were identified as neurons by dual immunofluorescent staining of GFAP and NeuN as markers of astrocytes and neurons, respectively. Then the dopaminergic neurons in AVPV were detected by immunohistochemistry staining of tyrosine hydroxylase (TH). No significant difference in the number of TH neurons was observed between the diet treatment groups. When estrogen receptor (ER) alpha and beta were examined by immunohistochemistry, we observed a 22% reduction of ERbeta-positive cell numbers in AVPV with consumption of soy isoflavones, whereas no significant change in ERalpha-positive cell numbers was detected. Furthermore, almost all the apoptotic cells were ERbeta-immunoreactive (ir), but not ERalpha-ir. Last, subcutaneous injections of equol (a major isoflavone metabolite) that accounts for approximately 70-90% of the total circulating plasma isoflavone levels did not alter the volume of AVPV in adult male rats.

Conclusion: In summary, these findings provide direct evidence that consumption of soy isoflavones, but not the exposure to equol, influences the loss of ERbeta-containing neurons in male AVPV.

Figure 1

Representative photomicrographs of apoptotic cells labeled by TUNEL staining in AVPV of 150 day-old male Long-Evans rats fed either an isoflavone-free diet (Phyto-free) or an isoflavone-rich diet (Phyto-600) from conception until time tissue collected. Apoptotic cells were visualized with diaminobenzadine (DAB) and seen as brown nuclear staining. Sections were counterstained with hematoxylin (blue). A and C are representative photomicrographs of the Phyto-free AVPV (n = 5). B and D are representative photomicrographs of the Phyto-600 AVPV (n = 5). Across the diet treatments, A corresponds to a similar coronal brain section in B. The AVPV is outlined with dashed lines. Boxed regions in A and B are magnified and shown in C and D, respectively. Significantly more apoptotic cells (arrows) were observed in Phyto-600 AVPV than the Phyto-free group (n = 5; p < 0.001; two-sample student t-test). Bar = 25 μm for all the photomicrographs.

Figure 2

Cell densities in AVPV of 150 day-old male Long-Evans rats fed either an isoflavone-free diet (Phyto-free) or an isoflavone-rich diet (Phyto-600) from conception until time tissue collected. Cell densities in (A) were measured from both apoptotic and non-apoptotic cells in the field of 10,000 μm², while the cell densities in (B) were measured from non-apoptotic cells. Each data point represents mean ± SEM of six measurements (two most condensed fields of three AVPVs out of five animals). * significantly greater cell density in the Phyto-600 fed male rats than the Phyto-free animals (p < 0.001; two-sample student t-test).

Figure 3

Representative photomicrographs of apoptotic cells (brown nuclear staining), astrocytes (red) and neurons (green) by TUNEL and dual GFAP/NeuN immunofluorescent staining in AVPV of 150 day-old male Long-Evans rats. All the photomicrographs (coronal brain sections) are from animals fed an isoflavone-rich diet (Phyto-600) from conception until time tissue was collected. An astrocyte (red; arrow) is indicated in A, whereas the neurons are displayed in green. B is a representative photomicrograph of TUNEL staining in AVPV (n = 5). C displays dual GFAP/NeuN immunofluoresent stained sections within AVPV, which is 6 μm apart from B. The apoptotic cells (arrows in B) were identified as neurons (arrows in C). Bar = 25 μm for all the photomicrographs.

Figure 4

Representative photomicrographs of immunohistochemistry for tyrosine hydroxylase (TH) labeled neurons in AVPV of 150 day-old Long-Evans rats fed either an isoflavone-free diet (Phyto-free) or an isoflavone-rich diet (Phyto-600) from conception until time tissue collected. TH-immunoreactive(ir) cells were visualized with diaminobenzadine (DAB) and seen as brown cytoplasma staining. Sections were counterstained with hematoxylin (blue). Positive control of TH staining is shown in Phyto-free fed female AVPV (A). In males, no significant differences in the number of TH-ir neurons were observed between Phyto-600 (C) and Phyto-free AVPV (B). The TH-ir cells in the boxed regions were indicated (*) at high magnification in the up-left corner of B and C. Bar = 25 μm for all the photomicrographs.

Figure 5

Representative photomicrographs of apoptotic cells (TUNEL) and ERalpha-immunoreactive cells (ERalpha IHC) in AVPV of 150 day-old male Long-Evans rats. All the photomicrographs (coronal brain sections) are from animals fed an isoflavone-rich diet (Phyto-600) from conception until time tissue collected. ERalpha-immunoreactive (ir) cells were visualized with diaminobenzadine (DAB) and seen as brown nuclear staining (arrows in ERalpha). Sections were counterstained with hematoxylin (blue). A, C, E, G and I are representative photomicrographs of TUNEL staining in AVPV (n = 5). B, D, F, H and J are ERalpha IHC stained sections within AVPV, which are 6 μm apart from A, C, E, G, and I, respectively. Apoptotic cells (stars in TUNEL) were not ERalpha-ir (stars in ERalpha). Bar = 25 μm for all the photomicrographs.

Figure 6

Representative photomicrographs of apoptotic cells (TUNEL) and ERbeta-immunoreactive cells (ERbeta IHC) in AVPV of 150 day-old male Long-Evans rats. All the photomicrographs (coronal brain sections) are from animals fed an isoflavone-rich diet (Phyto-600) from conception until time tissue collected. ERbeta-immunoreactive cells were visualized with diaminobenzadine (DAB) and seen as brown nuclear staining. Sections were counterstained with hematoxylin (blue). A, C and E are representative photomicrographs of TUNEL staining in AVPV (n = 5). B, D and F are ERbeta immunohistochemistry stained sections within AVPV, which are 6 μm apart from A, C and E, respectively. Except a few apoptotic cells (stars in C and E) missing in ERbeta IHC sections, all other apoptotic cells (arrows in TUNEL) were ERbeta-immunoreactive (arrows in ERbeta). Bar = 25 μm for all the photomicrographs.

Figure 7

Schematic diagram of the total number of cells relative to the numbers of ERbeta-ir cells and TUNEL stained cells in AVPV of Long-Evans rats fed either an isoflavone-free diet (Phyto-free) or an isoflavone-rich diet (Phyto-600) from conception until time tissue collected. The total number of cells in Phyto-free AVPV is similar to that of Phyto-600 (A). In the Phyto-free group, ERbeta-ir cells represent approximately 67.5% of the total number of cells (B), whereas TUNEL stained cells account for approximately 3.6% of total cells in AVPV (C). In the Phyto-600 group, ERbeta-ir cells represent approximately 52.8% of the total number of cells (B), whereas TUNEL stained cells account for approximately 33.1% of total cells in AVPV (C). Independent of diet treatment, most of the TUNEL stained cells express ERbeta.

Figure 8

Representative photomicrographs of ERalpha and ERbeta-immunoreactive cells (IHC staining) in AVPV of 150 day-old male Long-Evans rats. All the photomicrographs (coronal brain sections) are from animals fed an isoflavone-free diet (Phyto-free) from conception until time tissue collected. ER-immunoreactive (ir) cells were visualized with diaminobenzadine (DAB) and seen as brown nuclear staining. Sections were counterstained with hematoxylin (blue). A, C, E and G are representative photomicrographs of ERalpha IHC staining in AVPV (n = 2). B, D, F and H are ERbeta IHC stained sections within AVPV, which are 6 μm apart from A, C, E and G, respectively. Some cells are both ERalpha- and ERbeta-ir (stars for ERalpha and ERbeta). The number of cells in the AVPV that express both ERalpha and ERbeta is approximately 20–22% of the total number of cells within this nuclear structure. Some cells are only ERalpha-ir or ERbeta-ir (arrows in ERalpha or ERbeta). Bar = 25 μm for all the photomicrographs.

Figure 9

AVPV volumes of adult male Long-Evans rats after equol injection treatment. The volumes were measured and analyzed with Bioquant^®. No significant alterations were observed between equol-treated compared to the control (DMSO) group (two sample student t-test).