Forty-eight adult male and female UChA and UChB rats, aged 60 days (225-240 g), were obtained from the Department of Anatomy, Bioscience Institute/Campus of Botucatu, IBB/UNESP - Univ Estadual Paulista. The animals were randomly divided into two groups (n = 24/group). All animals were housed in polypropylene cages (43 cm × 30 cm ×15 cm) with laboratory-grade pine shavings as bedding and maintained under controlled temperature settings (23 ± 1°C) and lighting conditions (12-h L, 12-h D photoperiod, lights switched off at 0700 h). The animals were handled in accordance with the Ethical Principles in Animal Research adopted by the Brazilian College of Animal Experimentation (COBEA) and approved by the IBB/UNESP Ethical Committee for Animal Research, Protocol 01/08-CEEA.
After UCh rats were individually housed (aged 60 days), they were given a choice between two bottles containing either water or 10% (v/v) ethanol ad libitum for 15 days. After this period, 12 animals per group displaying ethanol consumption less than 1.9 g ethanol/kg BW/day (UChA strain) and higher than 2.0 g ethanol/kg BW/day (UChB strain; ranging from 4 to 5 g ethanol/kg/day) were selected for the experiment . For this study, the preference ratio associated with ethanol-seeking care was approximately 50%. In addition, to ensure better efficiency and prevent against damage arising from ethanol consumption during pregnancy, the animals were maintained without access to ethanol after preference determination.
Male and female rats (120 days old) derived from UChA and UChB lineages were allowed to mate. At night, mature females were housed together with males to encourage copulation. The confirmation of mating was seen in early morning by the presence of sperm on the slides. This finding was designated as day 0 of pregnancy. The rats were monitored once per day, and near the end of pregnancy, rats were monitored twice per day in order to determine time of pup's birth. The time and date of birth was fixed as the postnatal day 0 (PND 0), and the sexing and standardization in 8 pups/litter, with proper balance between male and female being ensured in order to avoiding any interference on maternal preference, also occurred at this time. During pregnancy and lactation, the following groups were formed: UChA mothers (n = 12) and UChB mothers (n = 12).
The mothers of both UCh lineages did not receive ethanol during mating, pregnancy or lactation in order to prevent the effects of Fetal Alcohol Syndrome.
Evaluation of maternal care
Mothers and offspring were housed in individual home-cages for evaluation of maternal care. The animals were monitored by one experimenter. Food and water were provided ad libitum. The maternal care was evaluated from birth (PND 0) until the 10th postnatal day (PND 10) during 60 minutes of observation four times a day. This observation amounted to 1056 h of observations. During the 60 minutes observations, each female was observed every 3 minutes for a total of 80 observations/mother/day. The observations occurred at regular times each day with three periods during the light phase (0800, 1200 and 1600 h) and one period during the dark phase (2000 h) [5, 7]. The measured categories of maternal care were adopted from previous work [28–31]: carrying, licking/grooming, arched-back nursing and licking/grooming, arched-back nursing, passive nursing and contact. No contact with the pups occurred when mothers were engaged in nest building, environmental exploration, self-grooming and feeding.
Estrous cycle analysis
At 90 days of age, the estrous cycles of female offspring were monitored by colpocytological examination (vaginal smears) every day for 21 consecutive days. Cells detaching from the vaginal epithelium were removed with a pipette (Lab Mate 0.5-10 μL, UK). Filter tips containing 10 μL 0.9% saline were discarded after the vaginal secretions had been transferred to clean slides . Colpocytological examination time was set at 0900 h. Each slide was analyzed under a Zeiss Axiophot II microscope (Carl Zeiss, Germany) at 10× and 25× magnifications.
Biological sample collection
At 120 days of age, all UChA and UChB rats in estrous were weighed and euthanized by decapitation. Blood samples were collected and stored at - 80°C for further analysis. Ovaries were dissected and weighed using analytical balance (Owalabor) and were fixed by immersion in 10% buffered formalin.
These analyses were performed using 5-μm-thick slices under light microscopy, adopting one section and discarding ten sections in sequence and finally resulting in 12 repetitions/ovary . The primordial, primary, growing (more than two layers until the antral cavity appear), preantral, antral and mature follicles were each counted. All data were analyzed under a Zeiss Axiophot II microscope (Carl Zeiss, Germany) using 20× magnification for primordial and primary follicles and 10X for others.
Immunohistochemistry for Ki-67
Sections of paraplast-embedded ovaries (5 μm) from each offspring were collected on silanized glass slides and pretreated with 2 N HCl for 30 min at 37°C. Antigen retrieval was achieved by incubating the slides with 0.1% trypsin for 15 min at 37°C. After washing, the slides were blocked with 3% hydrogen peroxide in methanol for 20 min and 3% bovine serum albumin (BSA) in PBS for 1 h at room temperature. Next, slides were incubated with monoclonal anti-Rat-Ki-67 antibody (clone MIB-5, Dako, Carpinteria, CA) at a 1:50 dilution in 1% BSA in PBS and incubated overnight at 4°C. After washing with PBS, the slides were incubated for 1 h at room temperature with biotinylated goat anti-mouse IgG antibody (Santa Cruz Biotechnology, CA) diluted 1:100 in 1% BSA in PBS. After washing, the sections were incubated with avidin-biotin-peroxidase solution (diluted 1:50) for 45 min (Elite ABC kit, Vector Laboratory, Burlingame, CA, EUA). Chromogen color development was carried out with 3,3'-diaminobenzidine tetrahydrochloride. Slides were counterstained with Harris's hematoxylin. A negative control was performed by omitting the primary antibody incubation step. The data were analyzed under a Zeiss Axiophot II microscope (Carl Zeiss, Germany). To quantitatively evaluate Ki-67-immunostained nuclei (proliferation index), the total number of positive granulosa cells in 10 randomly selected follicles was counted at 40× magnification for each follicular development stage. The results were expressed as a percentage of total cells counted (number of labeled nuclei × 100/total number of cells).
Western blotting analysis and protein quantification
After euthanasia, the ovaries from UCh offspring were rapidly removed, and tissue samples of 50 mg were immediately frozen in liquid nitrogen and stored at -80°C. All tissues were homogenized with RIPA lysis buffer (Pierce Biotechnology, Rockford, IL, USA), using a homogenizer (IKA® T10 basic Ultra, Staufen, Germany). Aliquots of 10% Triton were added to homogenates, and samples were placed on dry ice for 2 h for optimal extraction. These suspensions were centrifuged at 21,912 × g for 20 min at 4°C, and the pellet was discarded. Protein concentrations were measured by the Bradford method. Total proteins were dissolved in 1.5 × sample buffer previously described by Laemmli and used for SDS-PAGE (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of protein (70 μg) were loaded per well onto preformed gradient gels, 4-12% acrylamide (Amersham Biosciences, Uppsala, Sweden) with a Tris-glycine running buffer system for electrophoresis (60 mA fixed during 2 h). After electrophoresis, total proteins were electro-transferred (200 mA fixed by 1 h 30 min) onto 0.2 μm nitrocellulose membranes in a Tris-glycine-methanol buffer. Prestained standards were used as molecular weight markers. Thereafter, the membranes were blocked with TBS-T solution containing 3% BSA at room temperature (RT) for 60 min and subsequently incubated at 4°C overnight with rabbit primary antibody AR anti-androgen receptor (AR); rabbit clone E115 anti-ERα; and rabbit clone 68-4 anti-ERβ (dilutions of 1:1000; 1:250; 1:500 in 1% BSA, respectively). This step was followed by washing 3 × 5 min in TBS-T solution and incubation for 2 h at RT with rabbit HRP-conjugated secondary antibodies (diluted 1:1000 in 1% BSA; Sigma, St. Louis, MO, USA). After sequential washing with TBS-T, signals were enhanced by mixing 10 mL PBS, 8 μl H2O2 and 0.02 g diaminobenzidine (DAB) as chromogen. Immunoreactive bands of each protein were obtained from blots of six rats per group using image analysis software (NIS-Elements, Advanced Research, Nikon). β-actin was used as an endogenous control, and all results were expressed as means ± SEM. Immunoblotting concentrations were represented as optical densitometry values (band intensity/β-actin ratio).
Blood samples were collected into heparinized tubes from the trunk of decapitated UCh mothers and female offspring after variation of maternal care in the postpartum period. Afterwards, plasma was obtained by centrifugation at 1,200 × g for 15 min at 4°C and stored at -20°C until assayed by radioimmunoassay (RIA). Plasma samples were assayed for FSH and LH by double-antibody RIA with specific kits provided by the National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases (NIADDK, Baltimore, MD, USA). The FSH primary antibody was anti-rat FSH-S11, and the standard was FSH-RP2. The antiserum for LH was LH-S10 using RP3 as reference. The lower limit of detection for FSH and LH was 0.2 ng/mL and the intra-assay coefficients of variation were 3% and 4%, respectively. Plasma concentrations of E2 and P4 were determined using Estradiol and Progesterone Maia kits (Biochem Immunosystems, Serotec, Italy). The lower detection limit and the intra-assay coefficient of variation were, respectively, 7.5 pg/mL and 2.5% for E2 and 4.1 ng/mL and 3.7% for P4. Plasma concentrations of corticosterone were determined using specific kits provided by Sigma-Aldrich, Steinheim, Germany. Assay sensitivity was 0.023 ng/mL, and the intra-assay coefficient of variation was 4.5%.
All samples were measured in duplicate and at different dilutions when necessary. In order to prevent interassay variation, all samples were assayed in the same RIA.
The non-parametric Mann-Whitney test was used for general maternal care comparisons. Two Way Repeated Measures ANOVA was performed to evaluate the influence of time (days) along the period of observation (based on two independent factors: time and UCh varieties). Data of follicle counts and percentages were expressed as median followed by quartiles [Q1-Q3]. Student's t-test was applied to other parameters, and the results were expressed as means ± SEM. Differences were considered significant when p < 0.05. The statistical software used was GraphPad Instat version 4 and Sigma Plot version 11.0 for graphic design.