Long-Evans rats (12 males and 16 females) were purchased from Charles River Laboratories (Wilmington, MA, USA) at 50 days of age for breeding. These animals were caged individually and housed in the Brigham Young University Bio-Ag vivarium and maintained on an 11-hour dark and 13-hour light schedule (lights on 0600–1900). The use of animals and the methods of this study were approved by the Institute of Animal Care and Use Committee (IACUC) at Brigham Young University (BYU).
Upon arrival all animals were allowed ad libitum access to water and either a commercially available diet with high phytoestrogen levels (Harlan Teklad Rodent Diet 8604, Madison, WI, USA) containing approximately 600 ppm of soy isoflavones (referred to hereafter as the Phyto-600 diet), or a custom diet (Ziegler Bros., Gardner, PA, USA) containing approximately 10–15 ppm of soy isoflavones (referred to hereafter as the Phyto-free diet) . The content and nutrient composition of these diets is described in detail elsewhere . The diets were balanced and matched for equivalent percentage content of protein, carbohydrate, fat, amino acids, vitamins and minerals, etc . Circulating phytoestrogen serum levels from rats maintained on these diets have been reported previously by our laboratory using GC/MS analysis [2, 16, 18, 40]. The animals were time mated within their respective diets so that the offspring of these pairings would be exposed solely to either the Phyto-600 or Phyto-free diet.
Brain sample preparation
At approximately 150 days of age, the male offspring (n = 5) by diet treatment were deeply anesthetized with a mixture of ketamine/acepromazine (75/2.5 mg/kg IP) and transcardially perfused with isotonic saline and then 10% buffered formalin. The whole brain was immediately removed from the skull and stored in 10% buffered formalin for 14 days and 10% sucrose for one week before being embedded in paraffin. Coronal brain sections were prepared at 6 micrometers with a microtome. The AVPV was located by using landmarks such as the anterior commissure and third ventricle. Then sections on the exact same plane from different animals were processed for further comparisons between animals described below.
To detect apoptosis in AVPV, NeuroTACSTM II (a reagent kit for in situ detection of apoptosis in neural tissue; Cat # 4823-30-K) was purchased from Trevigen, Inc. (Gaithersburg, MD, USA). NeuroTACS II utilizes terminal deoxynucleotidyl transferase (TdT) to incorporate biotinylated nucleotides at the sites of DNA breaks which are characteristic of apoptosis. The deparaffinized slides were stained according to the manufacturer's direction. Briefly, brain sections were rehydrated in ethanols and permeablized. Endogenous peroxidase was inactivated by 3% hydrogen peroxide. The sections were incubated in a humidity chamber with labeling reaction at 37°C for 1 h, then with streptavidin HRP for 15 min at room temperature. Next they were developed with diaminobenzidine (DAB) and counterstained with hematoxylin. After dehydration, the slides were coversliped with Permount (Cat # 26905, Richard-Allan Scientific, Kalamazoo, MI, USA).
The samples were viewed with an Olympus BX61 microscope using 40× objectives. TUNEL-positive apoptotic cells exhibited a brown nuclear staining. The total number of counterstained cells in AVPV in a single section was counted (n = 5). Then, the total number of apoptotic cells in AVPV on the same section was recorded. Additionally, the total cumulative incidence of apoptosis was calculated as a percentage of the total population of cells. Then the cellular density of the AVPV was calculated by counting the cell number in 2 most condensed fields of 100 × 100 μm within AVPV in each section and expressed as number of cells per 10,000 μm2. For each group, a total of 6 measurements on cellular density were performed.
Dual immunofluorescent (IF) staining of GFAP and NeuN
After TUNEL staining, the adjacent sections were stained with dual immunofluorescent staining of GFAP and NeuN (the markers for astrocytes and neurons, respectively). GFAP (Glial Fibrillary Acidic Protein), the main constituent of intermediate filament of astrocytes, is found in the cytoplasm and appendages. NeuN (NEUronal Nuclei) is found only in neurons. The deparaffinized and rehydrated sections were microwaved in 10 mM Sodium Citrate buffer (antigen retrieval) at full power (900 W) for 1 min, followed by 9 min at half power and 20 min to cool down. After being blocked in 3% goat serum in PBS for 1 h, the sections were incubated in a humidity chamber at 4°C overnight with the primary antibodies, a rabbit anti-GFAP (1:1000, Cat # AB5804, Chemicon, Temecula, CA, USA) and a mouse anti-NeuN (1:100, Cat # MAB377, Chemicon, Temecula, CA, USA). The sections were then rinsed three times in phosphate buffered saline (PBS) for 5 min each and incubated in a humidity chamber for 1 h at room temperature with secondary antibodies, a far-red-fluorescent Alexa Fluor 633 dye labeled goat anti-rabbit IgG (1:200; Cat # A-21072, Molecular Probes, Eugene, OR, USA) and a bright green-fluorescent Alexa Fluor 488 dye labeled goat anti-mouse IgG (1:200; Cat # A-11070, Molecular Probes, Eugene, OR, USA). Then, the samples were rinsed in PBS three times for 5 min each and mounted with Fluoromount-G (Cat # 0100-01, Southern Biotechnology Associates, Inc., Birmingham, AL, USA). Dual-immunofluorescent specimens were analyzed at high power (60× objective lens; oil) with an Olympus FluoView FV300 confocal microscope (Minneapolis, MN, USA) using Blue Argon (488 nm) laser and Red Helium Neon (633 nm) laser.
Immunohistochemical (IHC) staining of tyrosine hydroxylase (TH)
As described above for immunofluorescent staining, the antigen was retrieved by incubation in 10 mM sodium citrate buffer and endogenous peroxidase was inactivated by 3% hydrogen peroxide. Then the samples were blocked in 3% goat serum in PBS and incubated at 4°C overnight in primary antibody solution of goat anti-TH (1:500, Cat # AB152, Chemicon, Temecula, CA, USA), which was localized with a biotinylated goat anti-rabbit IgG (1:500, Cat # AP132P, Chemicon, Temecula, CA, USA; 1 h at room temperature). Staining (DAB), counterstaining (hematoxylin), mounting and viewing was the same as described for TUNEL staining.
Immunohistochemical (IHC) staining of estrogen receptor (ER) alpha and beta
As described above for TUNEL and TH IHC staining, the sections were deparaffinized, rehydrated and permeablized, followed by the antigen retrieval in sodium citrate buffer and inactivation of endogenous peroxidase in 3% hydrogen peroxide. After being blocked in 3% goat serum in PBS for 1 h at room temperature, the sections were incubated in a humidity chamber at 4°C overnight with rabbit anti-ERalpha (1:1000, Cat # 06-935, Upstate, Lake Placid, NY, USA) or rabbit anti-ERbeta (1:100, 10 μg/ml, Cat # 06-629, Upstate, Lake Placid, NY, USA; these antibodies have been employed previously with validated methods [41, 42]. Immunoparticipate was visualized by an ABC Elite kit and DAB methods (Cat # PK-6101 and Cat # SK-4100, respectively, Vector Laboratories, Burlingame, CA, USA). Counterstaining (hematoxylin), mounting and viewing was the same as described for TUNEL staining.
ER-positive apoptotic cells exhibited a brown nuclear staining. The total number of counterstained cells in AVPV in a single section was counted (n = 5). Then, the total number of ER-positive cells in AVPV on the same section was recorded. Finally, the percentage of ER-positive cells of total cells in AVPV was calculated for each animal. Negative controls for all labeling studies were carried out by omitting TdT (TUNEL) or the primary antibodies.
Equol injection treatment
Male Long-Evans rats at 50 days of age were placed on the Phyto-free diet. At 150 days of age the rats were matched by body weight and then divided into control and equol treatment groups (n = 4). At 190 days, the rats received daily s.c. (0.1 cc) injections of control vehicle (DMSO) or equol at approx. 2.5 mg/Kg for 25 consecutive days. At 215 days of age the animals were sacrificed, blood was collected for equol levels; the brains processed via standard staining and analyzed via Bioquant® for morphometric AVPV parameters by treatments [2, 16]. The serum equol levels were equivalent to consuming Phyto-600 diet.
All the data were expressed as Mean ± SEM and were tested by 2-sample Student T-test in Minitab. Values were considered significantly different if p < 0.05.