231 CELLULAR PROLIFERATION IN FETAL OVARIAN FOLLICLES FROM LATE PREGNANT SHEEP FED MAINTENANCE OR RESTRICTED DIETS WITH NORMAL OR ENHANCED SELENIUM CONCENTRATIONS

Abstract

Hypertrophy and hyperplasia are the major processes for tissue growth and development. The fetal ovaries represent a type of tissue that expresses high cellular proliferation rates. Selenium (Se) is a mineral that has diverse biological functions and affects cellular proliferation in numerous tissues including cancer, digestive tract, and placenta. It has been demonstrated that levels and sources (organic vs. inorganic) of Se may affect tissue growth. This experiment was designed to determine whether maternal consumption of differing levels of energy and Se impacts cell proliferation in fetal ovarian follicles. Sheep (n = 36) were fed a maintenance (M; 2.12 Mcal/kg) or energy restricted (ER; 60% of maintenance; nutrition restriction occurred from Day 50 to Day 135 of pregnancy) diet with high Se (HSe; 81.5 �g/kg body weight) or normal Se (NSe; 7.4 �g/kg body weight) concentration from 21 days before breeding to Day 135 of pregnancy. At slaughter on Day 135 of pregnancy, fetal tissues were collected and fixed in Carnoy's solution. Ovaries (n = 3-6/treatment group) were weighed, sectioned (one section along the longitudal axis/ovary) and stained for the presence of proliferating cell nuclear antigen (PCNA), a marker for proliferating cells. To determine the proportion of proliferating primordial follicles or the labeling index (percentage of proliferating cells; Jablonka-Shariff et al. 1994 Biol. Reprod. 51, 531) for primary, secondary, and antral follicles, digital images of the tissues were taken and analyzed using a computerized image analysis program (Image-Pro Plus; Media Cybernetics, Inc., Silver Spring, MD, USA). The primordial follicle was considered as proliferating when at least one granulosa cell was PCNA-positive. The number of proliferating and non-proliferating cells was determined for granulosa of primary follicles (n = 225 total) and for granulosa and theca cells of secondary (n = 198) and antral (n = 96) follicles, and used to calculate the labeling index. The data were analyzed using the general linear models procedure of SAS (SAS Institute, Inc., Cary, NC, USA). The number of proliferating primordial follicles was decreased (P < 0.05) by restricted energy diet and Se treatment (11.9 � 1.7 for NSe-M diet vs. 7.2 � 1.3 for HSe-M diet and 8.3 � 0.8 for NSe-ER diet vs. 4.7 � 0.8 for HSe-ER diet). However, energy restriction or Se did not affect labeling index in primary, secondary, and antral follicles. The labeling index was similar for theca and granulosa cells from secondary, or antral follicles. The labeling index was greatest (P < 0.05) for antral, less for secondary and least for primary follicles (24.2 � 1.1% vs. 20.2 � 0.7% vs. 13.3 � 0.4%). These results demonstrate that both level of energy and Se in the maternal diet affects cellular proliferation in primordial but not in primary, secondary, or antral follicles in fetal ovaries. In addition, cellular proliferation increases as fetal follicular development progresses. These data indicate that level of energy and Se in the maternal diet may impact fetal ovarian development during the early stage of folliculogenesis. This work ws supported by USDA grant 2005-35206-15281 and Hatch Project ND01712.