Tag Archives: follicle (Part 2)

Methoxychlor-Induced Atresia: MATERIALS AND METHODS(11)

A standard curve was generated from five serial dilutions of one of the samples, thus allowing analysis of the amount of cDNA in the exponential phase. FSHR primer sequences were: (forward) 5′-AGCAAGTTTGGCTGTTATGAG G-3′, (reverse) 5′-GTTCTGGATGATTTAGAGG-3′. An initial incubation of 95°C for 10 min was followed by denaturing at 94°C for 10 sec, annealing at 55°C for 10 sec, and a final extension at 72°C for 10 sec for 50 cycles followed by final extension at 72°C for 10 min. A standard curve was generated by the software, and p-actin was used for each sample as an internal standard. Final values for FSHR expression were calculated as the ratio FSHR: p-actin. Primers specific for the mouse p-actin were used as an internal control as previously described. All experiments were performed in triplicate.

Methoxychlor-Induced Atresia: MATERIALS AND METHODS(10)

METHODS(10)

To ensure that proteins were loaded equally in each lane, the blots were stripped using ImmunoPure elution buffer (Pierce) and incubated with a p-actin (1:3000 dilution, Santa Cruz Biotechnology) and HRP-conjugated anti-mouse polyclonal antibody. Scanning densitometry was used to estimate the pixel density of individual bands using Molecular Analyst Software (Bio-Rad Laboratories, Hercules, CA).

Methoxychlor-Induced Atresia: MATERIALS AND METHODS(9)

All samples were run in duplicate. Sensitivity for the FSH assay was 200 pg/ml with inter- and intraassay coefficients of variation of 2.7% and 6.7% respectively. Sensitivity for the LH assay was 86 pg/ml with inter- and intraassay coefficients of variation of 5.3% and 2.5% respectively.

Methoxychlor-Induced Atresia: MATERIALS AND METHODS(8)

METHODS(8)

Follicle Stimulating Hormone and Luteinizing Hormone Assays

Blood was collected from CD-1 mice during estrus and FSH and LH assays were carried out by radioimmunoassay (RIA) using reagents from the National Hormone and Pituitary Distribution Program. For FSH, we conducted measurements at all doses (8-64 mg/kg MXC); however, insufficient blood remained to measure LH at all doses, so we chose to measure the control and 32 mg/kg dose, a dose at which we observed an effect of MXC on the ovary. Rat FSH and LH hormone antigen, rat FSH and LH antiserum, and mouse FSH and LH reference preparation were provided by the National Institute of Diabetes and Digestive and Kidney Diseases.

Methoxychlor-Induced Atresia: MATERIALS AND METHODS(7)

A blue filter was used on the microscope to filter out the blue counterstain of the sections. Standardized levels remained calibrated for all sections analyzed. Representative stained ovarian sections were chosen and three measurements of pixel density were made of the granulosa and thecal layers of every antral follicle in that section. For instance, in a section containing seven antral follicles, a total of 21 measurements of the granulosa cell layer and 21 measurements of the thecal layer were made. Means and standard error of the means were determined for pixel density measurements of granulosa and thecal layers from three individual ovarian sections of sesame control and 64-mg kg-1 day-1 MXC-treated mice.

Methoxychlor-Induced Atresia: MATERIALS AND METHODS(6)

METHODS(6)

In some experiments, sections were processed for immunolocalization of ERa and ERp using a commercially available antibody against ERa (1:80 dilution; Santa Cruz Biotechnology,) or ERp (1:360 dilution; Zymed Laboratories, San Franscisco, CA). The secondary antibody and visualization reagents were used according to the manufacturer’s instructions from the HistoMouse-SP Kit (Zymed Laboratories). As a control for background staining, two methods were used: incubation with secondary antibody, but no primary antibody, or incubation with normal rabbit serum followed by secondary antibody for confirmation of results. Both types of negative controls produced the same result. Six to eight tissue sections were analyzed per slide.

Methoxychlor-Induced Atresia: MATERIALS AND METHODS(5)

To estimate the number of corpora lutea (CL) per ovary, sections were used to count the number of CL without knowledge of treatment group. To avoid double counting, each CL was followed through consecutive sections to ensure that it was only counted once.

Methoxychlor-Induced Atresia: MATERIALS AND METHODS(4)

METHODS(4)

Ovarian sections were sampled and follicles were counted according to published methods. Briefly, a stratified sample consisting of every 10th section was used to estimate the total numbers of antral follicles per ovary. The selected sections from one ovary were randomized and the number of antral follicles was counted in the entire section. Only follicles with a visible nucleolus were counted to avoid double counting. Sections were counted without knowledge of treatment.

Methoxychlor-Induced Atresia: MATERIALS AND METHODS(3)

We elected to use the 64 mg/ kg MXC dose in the transgenic animal experiments because our experiments in CD-1 mice showed that this dose was the most effective in inducing atresia without overt toxicity. Transgenics and their wild-type counterparts were aged between 30 and 66 days at the start of the dosing experiments.

Methoxychlor-Induced Atresia: MATERIALS AND METHODS(2)

METHODS(2)

Females from these colonies between the ages of 30 and 66 days were housed (3-6 animals per cage) at the University of Maryland Central Animal Facility. Transgenic mice were genotyped using polymerase chain reaction (PCR)-based assays that have been previously described. Only wild-type mice and mice with homozygous deletion or overexpression were used in experiments. All animals were provided food and water for ad libitum consumption. Temperature was maintained at 22 ± 1°C and animals were subjected to 12L:12D cycles.

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