Sheep were fed a maintenance (M) diet with adequate (A) Se or high (H) Se concentration from 21 days before breeding to day 135 of pregnancy. From day 50 to day 135 of pregnancy (tissue collection day), a portion of the ewes from ASe and HSe groups were fed restricted (R; 60% of M) diet. Fetal ovarian sections were stained for: 1) the presence of proliferating cell nuclear antigen (a marker of proliferating cells) to determine the proportion of proliferating primordial follicles, or the labeling index (LI; percentage of proliferating cells) for primordial, primary, secondary and antral follicles, stromal tissues, and blood vessels; 2) factor VIII (a marker of endothelial cells) or 3) a presence of apoptotic cells/bodies. The number of proliferating primordial follicles and the LI of primordial follicles was decreased by R and/or HSe diets. The LI was similar for theca and granulosa cells, and for secondary or antral follicles, but was greater in secondary and antral than in primordial and primary follicles. R diet and/or Se affected the LI in all follicle types, in stromal tissues and blood vessels. A dense network of blood vessels was detected in the areas containing secondary to antral follicles, medulla, and hilus, but areas containing primordial follicles were poorly vascularized. The number of apoptotic cells was minimal. These results demonstrate that nutrient restriction and/or Se level in the maternal diet affected cellular proliferation in follicles, blood vessels, and stromal tissues in fetal ovaries. Thus, plane of nutrition and Se in the maternal diet may impact fetal ovarian development and function.

Ovine placentomes vary in shape, with type A placentomes being concave, type D convex, and types B and C intermediate in morphology. It has been speculated that as placentomes advance in type they differ in vascularity and nutrient transport capacity. Our objective was to determine cellularity and vascularity measurements, angiogenic factor expression, and arterial vasoactivity within different morphologic types of placentomes. On Day 130 of gestation, placentomes were collected from multiparous ewes (n = 38) and were evaluated for size, cellularity estimates, angiogenic factor mRNA expression, capillary vascularity (capillary size, capillary surface density [CSD], capillary number density [CND], and capillary area density [CAD]), and vasoreactivity to potassium chloride and angiotensin II. The average weight and size of type A and B placentomes were less (P < 0.01) than those of type C and D placentomes. Placentome morphology did not affect (P > or = 0.24) cotyledonary or caruncular cellularity estimates or percentage of cellular proliferation. Placentome morphology affected (P > or = 0.41) neither caruncular CAD, CND, CSD, or capillary size nor cotyledonary CND, CSD, or capillary size. Cotyledonary CAD was increased (P < 0.01) in type B and D placentomes compared with type A placentomes. Furthermore, placentome type did not affect (P > or = 0.06) angiogenic factor gene expression in the cotyledon or the caruncle. Size, but not morphologic type of placentome, was associated with greater caruncular artery contractility to potassium chloride and angiotensin II (P < 0.01 for both). Placentome size, but not morphologic type, may be important for vascularity and nutrient transfer in the placenta of the pregnant ewe.

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.

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