Materials and Methods Thirty female Sprague-Dawley rats weighing 180-210g were selected randomly and kept under constant conditions of light (12 h light/dark cycles) and temperature (21-24ºC). The experimental high throughput screening protocols and animal handling procedures were reviewed and approved by the Animal Ethics Committee of Shiraz University of Medical Sciences. Experimental Design The animals were randomly divided into six groups (n=5). The first group received 3 mg/kg (low dose) intraperitoneal (IP) administration

of ND (GMBH Hamburg Germany) weekly for four weeks. The second group received 10 mg/kg (high dose) for the same Inhibitors,research,lifescience,medical period of time. The third group received 0.1 ml IP administration of olive oil as vehicle (Darou Pakhsh Tehran Iran) for the same period of time (vehicle 1). The fourth and fifth groups received 10 IU of human menopausal gonadotropin (hMG) (Institut Biochimique SA-CH, Tehran, Inhibitors,research,lifescience,medical Iran) IP administration concurrent with the 3 mg/kg ND (low-dose hMG) and 10 mg/kg ND (high-dose hMG) for four weeks. The sixth group received olive oil and saline

in the same volume IP administration (vehicle 2). Vaginal smear was performed before every single injection to make sure that the animals were at the estrous phase of the estrous cycle. Inhibitors,research,lifescience,medical At the end of the experiment, blood samples were taken from the rats’ tails (1 mL) in order to determine the levels of FSH, LH, estrogen, and progesterone. Then, the rats were scarified Inhibitors,research,lifescience,medical using anesthetic Paclitaxel diethyl ether. The ovaries of each animal were removed and fixed in 10% formalin fixative for 24 h before they were dehydrated and embedded in paraffin. Stereological Methods The orientated method was used to obtain isotropic uniform random (IUR) sections.11 The paraffinized ovaries were sectioned serially in 5 µm thickness (H&E staining) for volume estimation and 25 µm thickness (Feulgen staining) for number. Estimating the Number of Primordial Follicle Morphological classification of the follicles in the rats was determined as primordial if a follicle contained an oocyte surrounded

by a partial or complete Inhibitors,research,lifescience,medical layer of squamous granulose cells. The number of primordial Entinostat follicles was determined using an optical disector design applied to 25 µm thick sections. This method is based on the direct counting of the particles (in this study, the oocyte nucleoli) in the original structure. The total numbers of the primordial follicles was estimated by stereological software. The unbiased counting frame was superimposed on the images that were viewed on the monitor (figure 1). An average of 80-100 microscopic fields were selected in each ovary via a systematic sample. The position of the first area was selected randomly outside the sections and the other areas were selected by moving the microscope stage in an equal interval along the x- and y- directions using a stage micrometer. A high numerical aperture (×100 magnification)-(NA=1.