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Skin fibroblasts from patients with diabetes mellitus display abnormalities in cell proliferation. The use of exogenous growth factors on diabetic wounds has been found to stimulate fibroblast proliferation and facilitate wound healing. However, the results of application of FGF-2 alone to diabetic wounds in clinical trials have been disappointing. The objective of this experiment was to study the effects of FGF-2 and media supplements on in vitro proliferation of skin fibroblasts from patients with type II diabetes and nondiabetic controls, and to evaluate the association between fibroblast proliferation and cAMP production. Fibroblast cell lines (n = 5 from diabetic and n = 5 from control individuals) were cultured in DMEM + 20% FBS for 7 days. Cells were then counted, plated into 24-well plates at a concentration of 2 x 10(4) cells/well and incubated for 24 h in DMEM with serum. The next day, medium was changed to serum-free DMEM alone or DMEM with supplements (albumin, transferrin, insulin and hydrocortisone). Cells were cultured in the presence or absence of varying doses of FGF-2 (0, 0.3, 1, 3, 10 and 30 ng/ml) for 72 hrs then counted and medium was collected for cAMP radioimmunoassay. The doubling time for cell number tended to be greater (p < 0.2) for diabetic fibroblasts than for control fibroblasts. The addition of supplements to the medium reduced (p < 0.05) the doubling time for both fibroblast types. FGF-2 stimulated (p < 0.05) proliferation of diabetic fibroblasts only in medium containing supplements. In contrast, FGF-2 stimulated proliferation of control fibroblasts in medium with or without supplements. The maximal effects of FGF-2 on fibroblast proliferation were greater (p < 0.02) in medium with supplements than in medium without supplements. The K(D) of FGF-2 for fibroblast proliferation was greater (p < 0.06) for diabetic than for control fibroblasts, and lower (p < 0.02) for medium with supplements than for medium without supplements. Fibroblasts from patients with diabetes mellitus produced more (p < 0.05) cAMP than control fibroblasts. These results demonstrate that FGF-2 requires the presence of supplements to enhance proliferation of fibroblasts from patients with type II diabetes mellitus. In addition, fibroblasts from diabetic patients showed a greater K(D) for FGF-2 in terms of cell proliferation. These data suggest a defective FGF receptor or down-regulation of the FGF receptor-mediated cascade that leads to cell proliferation. Identifying methods of reducing the K(D) of FGF-2 in stimulating the proliferation of diabetic fibroblasts may improve the clinical response of diabetic wounds to FGF-2.

To characterize mitogenic factors produced by ovine endometrium during early pregnancy, endometrial explant-conditioned media (ECM) were obtained from ewes on day 12, 18, 24, or 30 after mating. These ECM contained mitogenic activity for both endothelial and 3T3 cells across all days. The endothelial mitogenic activity was greatest on day 24, whereas mitogenic activity for 3T3 cells did not differ across days. By ultrafiltration, ion exchange, and heparin-affinity chromatography, the endothelial mitogenic activity was found to have a molecular mass greater than 100 kDa, to be anionic, and to be heparin binding, respectively. Three peaks of endothelial mitogenic activity were recovered from heparin-affinity chromatography. The major peak, H3, was mitogenic for endothelial but not for 3T3 cells. H3 was further purified, and the single peak of heparin-binding activity, designated H3b, represented a 681-fold purification of endothelial mitogenic activity from endometrial ECM. H3 and H3b were heat labile and trypsin sensitive, and their biological activity was heparin enhanced. The majority of the endothelial mitogenic activity was immunoneutralized by antibodies against acidic and basic FGF. Nevertheless, we were unable to detect bFGF in H3 or H3b by Western immunoblot analysis. Thus, in this study we have extended our previous observations and demonstrated that (i) during early pregnancy the ovine endometrium produces mitogenic activity for both endothelial and 3T3 cells, (ii) the endothelial mitogenic activity is greatest on day 24 after mating, which corresponds with the onset of endometrial vascular growth, and (iii ) the major endothelial mitogen has a high affinity for heparin, and although it is immunologically related to FGF, it differs from known FGF in its apparent molecular size and biological activities.Key words: heparin-binding growth factor, fibroblast growth factor, uterus, early pregnancy, ewe.

Objectives were to investigate effects of nutritional plane and Se supply during gestation on visceral organ mass and intestinal growth and vascularization in ewes at parturition and during early lactation. Primiparous Rambouillet ewes (n = 84) were allocated to 2 × 3 × 2 factorial arrangement of treatments. Factors included dietary Se [adequate Se (ASe, 11.5 μg/kg BW) or high Se (HSe, 77.0 μg/kg BW)], nutritional plane [60% (restricted; RES), 100% (control; CON), or 140% (high; HIH)], and physiological stage at necropsy (parturition or d 20 of lactation). At parturition, lambs were removed and 42 ewes (7 per treatment) were necropsied. Remaining ewes were transitioned to a common diet which met lactation requirements and mechanically milked for 20 d. In the absence of interactions (P0.10), main effects are reported. At parturition, stomach complex and liver masses were greatest for HIH, intermediate for CON, and least for RES (P0.02). Small intestinal mass was greater (P ≤ 0.002) for HIH than RES and CON, and greater (P0.01) for ASe than HSe. During early lactation, RES and CON gastrointestinal masses increased disproportionally to BW (P0.05). At parturition, jejunal mucosal density was less (P ≤ 0.01) for RES than CON and HIH, whereas CON had greater (P0.003) jejunal mucosal RNA concentration and RNA:DNA than RES and HIH. Although there were no differences (P0.17) at parturition, jejunal cell percent proliferation was greatest in RES, intermediate in CON, and least in HIH (P ≤ 0.09) at d 20 lactation. At both stages, RES had less (P = 0.01) jejunal capillary area density than HIH and less (P ≤ 0.03) capillary surface density than CON and HIH. During lactation, jejunal capillary size was greater (P = 0.04) for ewes previously fed HSe compared with ASe. At parturition, ASe-HIH had greater (P0.02) jejunal mucosal endothelial nitric oxide synthase 3 mRNA than all other treatments and greater (P = 0.10) vascular endothelial growth factor (VEGF) than all treatments, except ASe-RES. In addition, CON had less (P ≤ 0.08) jejunal VEGF receptor-1 (FLT1) mRNA compared with RES and HIH, and ASe had greater (P = 0.003) FLT1 than HSe at parturition. Ewes fed HIH had greater (P = 0.04) jejunal VEGF receptor-2 mRNA compared with RES. Results indicate that maternal intestinal growth and vascularization are responsive to nutritional plane and dietary Se during gestation and undergo changes postpartum when under similar lactational management.

The effects of maternal nutrition on offspring wool production (quality and quantity) were evaluated. Primiparous Rambouillet ewes (n = 84) were randomly allocated to 1 of 6 treatments in a 2 × 3 factorial design. Selenium treatment [adequate Se (ASe, 9.5 μg/kg of BW) vs. high Se (HSe, 81.8 μg/kg of BW)] was initiated at breeding, and maternal nutritional intake [control (CON, 100% of requirements) vs. restricted (60% of CON) vs. overfed (140% of CON)] was initiated at d 50 of gestation. Lamb birth weight was recorded at delivery, and all lambs were placed on the same diet immediately after birth to determine the effects of prenatal nutrition on postnatal wool production and follicle development. At 180 ± 2.2 d of age, lambs were necropsied and pelt weights were recorded. Wool samples were collected from the side and britch areas, whereas skin samples were collected from the side of each lamb only. Although Se status did not influence side staple length in males, female lambs born from ewes on the ASe treatment had a shorter staple length (P0.05) when compared with females from ewes on the HSe treatment. Maternal nutritional intake and Se status did not influence (P ≥ 0.23) wool characteristics on the britch. However, at the britch, wool from female lambs had a reduced comfort factor (P = 0.01) and a greater (P = 0.02) fiber diameter compared with wool from male lambs. Maternal Se supplementation, maternal nutritional plane, sex of the offspring, or their interactions had no effect (P0.13) on primary (29.10 ± 1.40/100 µm(2)) and secondary (529.84 ± 21.57/100 µm(2)) wool follicle numbers. Lambs from ASe ewes had a greater (P = 0.03) secondary:primary wool follicle ratio compared with lambs from HSe ewes (20.93 vs. 18.01 ± 1.00). Despite similar postnatal diets, wool quality was affected by maternal Se status and the maternal nutritional plane.

The objectives were to investigate effects of nutritional plane and Se supply during gestation on yield and nutrient composition of colostrum and milk in first parity ewes. Rambouillet ewe lambs (n = 84, age = 240 ± 17 d, BW = 52.1 ± 6.2 kg) were allocated to 6 treatments in a 2 × 3 factorial array. Factors included Se [adequate Se (ASe, 11.5 µg/kg of BW) or high Se (HSe, 77.0 µg/kg of BW)] initiated at breeding, and nutritional plane [60 (RES), 100 (CON), or 140% (HIH) of requirements] initiated at d 40 of gestation. Ewes were fed individually from d 40, and lambs were removed at parturition. Colostrum was milked from all ewes at 3 h postpartum, and one-half of the ewes (n = 42) were transitioned to a common diet meeting lactation requirements and mechanically milked for 20 d. Colostrum yield was greater (P = 0.02) for HSe ewes than ASe, whereas CON had greater (P0.05) colostrum yield than RES and HIH. Colostrum Se (%) was greater (P0.01) for HSe than ASe. Colostrum from ewes fed HSe had less (P = 0.03) butterfat (%), but greater (P ≤ 0.05) total butterfat, solids-not-fat, lactose, protein, milk urea N, and Se than ASe. Colostrum from HIH ewes had greater (P ≤ 0.02) solids-not-fat (%) than RES, whereas RES had greater (P ≤ 0.04) butterfat (%) than CON and HIH. Colostrum from ewes fed the CON diet had greater (P = 0.01) total butterfat than HIH. Total solids-not-fat, lactose, and protein were greater (P0.05) in colostrum from CON than RES and HIH. Ewes fed HSe had greater (P0.01) milk yield (g/d and mL/d) than ASe, and CON and HIH had greater (P0.01) yield than RES. Milk protein (%) was greater (P ≤ 0.01) in RES compared with CON or HIH. Ewes fed HSe had greater (P0.01) milk Se (µg/g and mg/d) than ASe on each sampling day. Milk from CON and HIH ewes had greater (P0.01) total solids-not-fat, lactose, protein, and milk urea N than RES. Total Se was greater (P = 0.02) in milk from ewes fed the CON diet compared with RES. Somatic cell count and total somatic cells were greater (P ≤ 0.05) in milk from CON than RES. A cubic effect of day (P ≥ 0.01) was observed for milk yield (g and mL). Butterfat, solids-not-fat, lactose, milk urea N, and Se concentration responded quadratically (P ≤ 0.01) to day. Protein (%), total butterfat, and total Se, and somatic cells (cells/mL and cells/d) decreased linearly (P0.01) with day. Results indicate that gestational nutrition affects colostrum and milk yield and nutrient content, even when lactational nutrient requirements are met.

GRAZUL-BILSKA, A.T., REDMER, D.A., KILLILEA, S.D., ZHENG, J., and REYNOLDS, L.P. 1993. Initial characterization of endothelial mitogens produced by bovine corpora lutea from the estrous cycle. Biochem. Cell Biol. 71: 270-277. To further characterize mitogenic factor(s) present in luteal extracts or luteal explant conditioned media (LCM), bovine corpora lutea (CL) were homogenized or incubated in explant culture, respectively. After evaluation of luteal extracts and LCM by using an endothelial cell proliferation bioassay, mitogenic activity was characterized by immunoneutralization with antibodies against heparin-binding (fibroblast) growth factor (HBGF) 1 or 2. LCM also were subjected to ultrafiltration, as well as anion-exchange, cation-exchange, and heparin-affinity chromatography. The presence of HBGF-2 in LCM also was evaluated by using a dot immunoblot assay. Extracts of luteal tissues and LCM stimulated (P < 0.05) proliferation of endothelial cells in a dose-dependent manner. Mitogenic activity of luteal extracts and LCM was decreased (P < 0.05) by treatment with specific antibodies against HBGF-2 or HBGF-1. LCM also contained immunoreactive HBGF-2. The mitogenic activity bound to anion exchangers, phenyl-Sepharose, and heparin-agarose, but not to cation exchangers, indicating that endothelial mitogenic activity is anionic at neutral pH, has some hydrophobic characteristics, and belongs to the HBGF family of proteins. Following ultrafiltration, endothelial mitogenic activity was retained by membranes having a 30 000 or 100 000 molecular weight cutoff. In addition, LCM was resolved into four peaks of heparin-binding endothelial mitogenic activity, each with a different affinity for heparin. These data demonstrate that bovine CL contain and produce endothelial mitogens of large molecular size, which may be important regulators of luteal function. These endothelial mitogens are heparin-binding and anionic at neutral pH. In addition, a large portion of the endothelial mitogenic activity produced by bovine CL appears to be immunologically related to HBGF-2 (basic fibroblast growth factor).

To determine the effects of maternal Se intake and plane of nutrition during mid or late gestation or both on AA concentrations and metabolite concentrations in the dam and fetus, pregnant ewe lambs (n = 64) were assigned to 1 of 8 treatments arranged in a 2 × 2 × 2 factorial array: Se level [initiated at breeding; adequate (ASe; 3.05 μg/kg of BW) or high (HSe; 70.4 μg/kg of BW)] and nutritional level [100% (control; CON) or 60% (restricted; RES) of NRC recommendations] fed at different times of gestation [d 50 to 90 (mid) or d 91 to 132 (late)]. A blood sample was obtained from each ewe and fetus on d 132 of gestation and used to measure circulating concentrations of glucose, NEFA, blood urea N, and AA. The late RES ewes and their fetuses had less (P ≤ 0.03) circulating glucose compared with late CON ewes and fetuses at d 132; however, no effect (P ≥ 0.14) of diet on the fetal:maternal glucose concentration ratio was observed. Late RES ewes had a smaller (P = 0.01) fetal:maternal NEFA ratio compared with late CON ewes. Ewes fed ASe had a greater (P = 0.01) fetal:maternal blood urea N ratio compared with HSe ewes. Fetal:maternal ratios of total circulating AA, total essential AA, and total nonessential AA were each affected (P ≤ 0.03) by the combination of Se treatment and late gestation nutritional level.

Lambs born from feed-restricted or overfed ewes can be lighter at birth, whereas maternal Se supplementation can increase fetal size near term. We hypothesized that birth weight would be inversely related to feed efficiency and growth rates during postnatal development. To examine the effects of maternal dietary Se and nutrient restriction or excess on postnatal lamb growth, diet digestibility, and N retention, 82 ewe lambs (52.2 ± 0.8 kg) were allotted randomly to 1 of 6 treatments in a 2 × 3 factorial arrangement. Factors were dietary Se [adequate Se (9.5 μg/kg of BW; ASe) vs. high Se (Se-enriched yeast; 81.8 μg/kg of BW; HSe)] and maternal nutritional intake [60% (restricted, RES), 100% (control, CON), or 140% (high, HI) of NRC requirements]. Selenium treatments began at breeding. Nutritional treatments began on d 50 of gestation. Lambs were immediately removed from their dams at parturition, provided artificial colostrum, and fed milk replacer until weaning. After weaning, lambs were maintained using common management and on common diets until necropsy at 180 d. Male and female lambs from RES-fed ewes were lighter (P ≤ 0.03) at birth than lambs from CON-fed ewes, with lambs from HI-fed ewes being intermediate. Although maternal nutritional intake influenced (P0.06) BW gain before weaning on d 57, both maternal nutritional intake and sex of offspring influenced (P ≤ 0.09) BW gain from d 57 to 180. Although maternal nutritional intake did not influence (P ≥ 0.35) female lamb BW gain, male lambs from RES-fed ewes were lighter (P ≤ 0.09) than those from CON-fed ewes until d 162. By d 180, male lambs from RES- and HI-fed ewes were lighter (P ≤ 0.09) than those from CON-fed ewes. In a subset of lambs used in a feed efficiency study, namely, those born to ASe ewes, HI maternal nutritional intake decreased (P ≤ 0.09) ADG and G:F compared with lambs born to RES- and CON-fed ewes, which did not differ (P ≥ 0.60). Conversely, when lambs were born to HSe ewes, HI maternal nutritional intake increased (P ≤ 0.01) ADG and G:F compared with CON, with RES being intermediate. Moreover, lambs born to ASe-HI ewes had decreased (P0.01) ADG and G:F compared with lambs born to HSe-HI ewes. Furthermore, male lambs had a greater (P0.01) G:F than female lambs. Maternal diet did not affect (P ≥ 0.11) N retention in male lambs. These data indicate that maternal nutrition during gestation and sex of the offspring alter postnatal growth and efficiency of growth in offspring despite similar postnatal management.

The objectives of this study were to evaluate effects of maternal dietary restriction and Se supply on angiogenic factor mRNA expression in intestinal and mammary tissues, and jejunal crypt cell proliferation and vascularity in late-term fetal intestines. In Exp. 1, pregnant ewe lambs (n = 32; initial BW = 45.6 +/- 2.3 kg) were allotted randomly to 1 of 4 treatments. Treatments (initiated d 50 +/- 5 of gestation) were control (3.5 microg of Se.kg of BW(-1).d(-1)), Se-wheat (75 microg of Se.kg of BW(-1).d(-1)), selenate (Se3; providing 75 microg of Se.kg of BW(-1).d(-1)), selenate (Se15; providing 375 microg of Se.kg of BW(-1).d(-1)). Diets (DM basis) were similar in CP (15.5%) and ME (2.68 Mcal/kg). In Exp. 2, pregnant ewe lambs (n = 36; initial BW 53.8 +/- 1.3 kg) were allotted randomly to treatments in a 2 x 2 factorial arrangement. Factors were nutrition (control, 100% of requirements vs. restricted nutrition, 60% of controls) and dietary Se (adequate Se; 6 microg of Se.kg of BW(-1).d(-1) vs. high Se; 80 microg of Se.kg of BW(-1).d(-1)). Selenium treatments were initiated 21 d before breeding, and nutritional treatments were initiated on d 64 of gestation. Diets (DM basis) were 16% CP and 2.12 Mcal/kg of ME. In Exp. 1, Se15 increased (P = 0.07) vascular endothelial growth factor (VEGF) mRNA expression, whereas Se supplementation decreased (P = 0.06) kinase insert domain receptor (KDR) mRNA in maternal mucosal scrape on d 134 of gestation. Expression of VEGF mRNA was decreased by Se (P = 0.10) in fetal jejunum. In mammary tissue, fms-related tyrosine kinase 1 and KDR mRNA were greater in Se-wheat compared with Se3, and KDR expression was increased (P = 0.10) in Se15 compared with Se3. In Exp. 2, dietary restriction increased (Por = 0.07) expression of mRNA for VEGF, fms-related tyrosine kinase 1, KDR, neuropilin 1, neuropilin 2, and hypoxia-inducible factor 1, alpha subunit in mucosal scrapes from maternal jejunum. In fetal jejunum, soluble guanylate cyclase, was decreased (P = 0.01) by maternal dietary restriction from d 64 to 135 of gestation. Total microvascularity in fetal jejunum was reduced (P = 0.002) by maternal dietary restriction. Mammary gland expression of VEGF, neuropilin 1, angiopoietin receptor (endothelial tyrosine kinase), and endothelial nitric oxide synthase 3 increased (Por = 0.09), whereas angiopoietin 1 decreased (P = 0.05) due to nutrient restriction. Data indicate that expression of angiogenic factors and receptors in maternal intestine, mammary gland, and fetal jejunum are responsive to maternal nutrition and likely explain observed changes in tissue vascularity.

To investigate the effects of nutritional plane and Se supply during gestation on ewe and offspring performance and body composition, 84 Rambouillet ewe lambs (age = 240 +/- 17 d, BW = 52.1 +/- 6.2 kg) were allocated to a 2 x 3 x 2 factorial arrangement of treatments. Factors included Se [adequate Se (ASe, 11.5 microg/kg of BW) or high Se (HSe, 77.0 microg/kg of BW)] initiated at breeding, nutritional plane [60% (restricted, RES), 100% (control, CON), or 140% (high, HIH) of NRC requirements] initiated at d 40 of gestation, and physiological stage at necropsy [3 to 24 h postpartum or d 20 of lactation]. Ewes were fed and housed individually in a temperature-controlled facility. At parturition, all lambs were removed and artificially reared until necropsy on d 20.6 +/- 0.9 of age. Ewes assigned to the treatment at d 20 of lactation were transitioned to a common diet meeting lactation requirements and were mechanically milked. From d 95 of gestation through parturition and d 20 of lactation, ewe BW and BCS were least (Por= 0.01) in the RES treatment, intermediate in the CON treatment, and greatest in the HIH treatment. Ewes fed HSe had a greater (Por= 0.05) BCS increase than those fed ASe during mid- and late gestation. During gestation, ewes in the HIH treatment had the greatest (P0.001) ADG and G:F, those in the CON treatment were intermediate, and those in the RES treatment were least, whereas ewes fed HSe had greater (P0.001) ADG and G:F than those fed ASe during midgestation. Ewe backfat and LM area on d 135 of gestation were least (P0.001) in the RES treatment, intermediate in the CON treatment, and greatest in the HIH treatment, with ewes fed HSe having greater (Por= 0.03) backfat than those fed ASe. During the first 20 d of lactation, ewes fed the RES diet had greater (P0.09) G:F than those fed the CON and HIH diets. Physiological stage had no effect on ewe omental and mesenteric fat or perirenal fat weights, although both were greater (P0.001) for ewes fed the HIH diet than for those fed the RES and CON diets. At birth, lambs born to ewes in the RES group weighed less and had decreased curved crown rump lengths (P = 0.08) compared with those born to ewes in the CON and HIH groups, and lambs from ewes in the ASe-RES treatment were lighter (P0.08) than those from ewes in the HSe-RES, ASe-CON, and ASe-HIH treatments. Lambs from dams in the RES group had less (P0.05) BW from d 7 to 19 and decreased (P0.07) overall ADG compared with lambs from dams in the CON and HIH groups. Additionally, lambs from dams in the RES group had less (Por= 0.08) perirenal fat than their counterparts, and lambs from dams in the HIH group had greater (P = 0.01) omental and mesenteric fat than lambs from dams in the RES group. Postpartum serum Se of ewes and lambs (birth and d 19) was increased (P0.001) by HSe feeding during gestation. Results indicate that BW differences in pregnant ewes attributable to nutritional plane are accompanied by changes in body composition and offspring BW, both of which may be affected by Se supply.

Developmental programming refers to the programming of various bodily systems and processes by a stressor of the maternal system during pregnancy or during the neonatal period. Such stressors include nutritional stress, multiple pregnancy (i.e., increased numbers of fetuses in the gravid uterus), environmental stress (e.g., high environmental temperature, high altitude, prenatal steroid exposure), gynecological immaturity, and maternal or fetal genotype. Programming refers to impaired function of numerous bodily systems or processes, leading to poor growth, altered body composition, metabolic dysfunction, and poor productivity (e.g., poor growth, reproductive dysfunction) of the offspring throughout their lifespan and even across generations. A key component of developmental programming seems to be placental dysfunction, leading to altered fetal growth and development. We discuss various large animal models of developmental programming and how they have and will continue to contribute to our understanding of the mechanisms underlying altered placental function and developmental programming, and, further, how large animal models also will be critical to the identification and application of therapeutic strategies that will alleviate the negative consequences of developmental programming to improve offspring performance in livestock production and human medicine.

To determine the effects of maternal Se intake and plane of nutrition during midgestation, late gestation, or both on hormone and metabolite concentrations in the dam and on placental characteristics, pregnant ewe lambs (n = 64) were assigned to 1 of 8 treatments arranged in a 2 x 2 x 2 factorial array: Se level [initiated at breeding; adequate (3.05 microg/kg of BW) or high (70.4 microg/kg of BW)] and nutritional level [100% (control) or 60% (restricted) of NRC recommendations] fed at different times of gestation [d 50 to 90 (midgestation) or d 91 to 130 (late gestation)]. The control ewes had a greater (P = 0.01) percentage change in BW from d 50 than restricted ewes during both mid- and late gestation. Although blood urea N was not affected by either Se or nutritional level, restricted ewes had greater (P = 0.01) concentrations of circulating Se on d 66, 78, 106, 120, and 130 of gestation compared with control ewes. Both Se and timing of the nutritional level affected circulating progesterone; however, only nutritional level affected thyroxine and triiodothyronine concentrations in the dam. Nutrient restriction during late gestation decreased (Por= 0.01) fetal BW and fetal fluid weight compared with the control ewes (3.75 vs. 4.13 +/- 0.10 kg and 1.61 vs. 2.11 +/- 0.11 kg). Although neither Se nor nutritional level affected (Por= 0.1) placental, caruncular, or cotyledonary weights, cotyledonary cellular proliferation was decreased (P0.05) in ewes receiving a high concentration of Se compared with those receiving adequate Se. In addition, either Se or nutritional level affected vascular endothelial growth factor (VEGFA), VEGFA-receptor 1, VEGFA-receptor 2, and NO synthase mRNA abundance in the cotyledonary tissue. In the caruncular tissue, either Se or nutritional level affected VEGFA-receptor 1, placental growth factor, and NO synthase mRNA abundance. Selenium supplementation and the duration or timing of nutrient restriction appear to influence the endocrine and metabolic status of the ewe, which may influence nutrient transport and placental function.

The objective was to describe the effects of dietary Se concentration and source on fetal and maternal Se load when fed to nulliparous ewes during mid- to late pregnancy. Pregnant, nulliparous ewes (n = 32; 45.6 +/- 2.3 kg; 330 +/- 17 d of age) were randomly assigned to treatment diets. Treatments were 3.5 microg of Se.kg of BW(-1) x d(-1) from the basal Se in the diet (C1X); 75 (S20X) and 350 (S100X) microg of Se.kg of BW(-1) x d(-1), with additional Se from supplemental sodium selenate; and 75 microg of Se.kg of BW(-1) x d(-1), with additional Se from naturally occurring Se-enriched wheat grain (W20X). Treatment diets were initiated at d 50 of pregnancy and continued until slaughter at d 134 +/- 4 of pregnancy. Plasma samples were collected from the ewes immediately before treatments commenced and every 14 d for 70 d. At slaughter, plasma was collected from ewes and their fetuses. Ewes were randomly assigned to 1 of 8 consecutive slaughter days. Maternal and fetal LM, kidney, and liver samples were collected and stored. Tissue and plasma samples were analyzed for Se. Compared with other treatments, S100X resulted in the greatest maternal tissue and plasma Se loads (P0.001). However, based on the total amount of Se consumed during the treatment period, efficiency of Se loading was greatest for the W20X treatment. Compared with C1X and S20X, Se loading in fetal tissues and plasma was greater (P0.01) for S100X and W20X treatments. In S100X-treated ewes, maternal plasma Se increased rapidly from d 50 to 64 but remained unchanged thereafter. Maternal plasma Se increased steadily throughout the experiment in W20X and S20X ewes, but remained unchanged in C1X throughout the study. Sodium selenate fed at 350 microg of Se.kg of BW(-1) x d(-1) and Se-enriched wheat grain at 75 microg of Se.kg of BW(-1) x d(-1) to nulliparous pregnant ewes neither induced signs of selenosis nor negatively influenced ewe or fetal growth and development. Therefore, ewes in this study were capable of consuming greater than twice the current Se maximum tolerable limit as sodium selenate without experiencing selenosis. Selenium from Se-enriched wheat grain treatment seemed to cross the placenta to the fetus at greater efficiency than did Se from sodium selenate and was equivalent in Se-loading potential to sodium selenate-Se that was fed at nearly 5 times the amount of wheat grain Se.

The objectives were to examine effects of dietary Se supplementation and nutrient restriction during defined periods of gestation on maternal adaptations to pregnancy in primigravid sheep. Sixty-four pregnant Western Whiteface ewe lambs were assigned to treatments in a 2 x 4 factorial design. Treatments were dietary Se [adequate Se (ASe; 3.05 microg/kg of BW) vs. high Se (HSe; 70.4 microg/kg of BW)] fed as Se-enriched yeast, and plane of nutrition [control (C; 100% of NRC requirements) vs. restricted (R; 60% of NRC requirements]. Selenium treatments were fed throughout gestation. Plane of nutrition treatments were applied during mid (d 50 to 90) and late gestation (d 90 to 130), which resulted in 4 distinct plane of nutrition treatments [treatment: CC (control from d 50 to 130), RC (restricted from d 50 to 90, and control d 90 to 130), CR (control from d 50 to 90, and restricted from d 90 to 130), and RR (restricted from d 50 to 130)]. All of the pregnant ewes were necropsied on d 132 +/- 0.9 of gestation (length of gestation approximately 145 d). Nutrient restriction treatments decreased ewe ADG and G:F, as a result, RC and CR ewes had similar BW and maternal BW (MBW) at necropsy, whereas RR ewes were lighter than RC and CR ewes. From d 90 to 130, the HSe-CC ewes had greater ADG (Se x nutrition; P = 0.05) than did ASe-CC ewes, whereas ADG and G:F (Se x nutrition; P = 0.08) were less for HSe-RR ewes compared with ASe-RR ewes. The CR and RR treatments decreased total gravid uterus weight (P = 0.01) as well as fetal weight (P = 0.02) compared with RC and CC. High Se decreased total (g; P = 0.09) and relative heart mass (g/kg of MBW; P = 0.10), but increased total and relative mass of liver (Por = 0.05) and perirenal fat (Por = 0.06) compared with ASe. Total stomach complex mass was decreased (P0.01) by all the nutrient restriction treatments, but was reduced to a greater extent in CR and RR compared with RC. Total small intestine mass was similar between RC and CC ewes, but was markedly reduced (P0.01) in CR and RR ewes. The mass of the stomach complex and the small and large intestine relative to MBW was greater (P = 0.01) for RC than for CR ewes. Increased Se decreased jejunal DNA concentration (P = 0.07), total jejunal cell number (P = 0.03), and total proliferating jejunal cell number (P = 0.05) compared with ASe. These data indicate that increased dietary Se affected whole-body and organ growth of pregnant ewes, but the results differed depending on the plane of nutrition. In addition, the timing and duration of nutrient restriction relative to stage of pregnancy affected visceral organ mass in a markedly different fashion.

Peripubertal ewe lambs (44.3 +/- 1.1 kg of initial BW) were used in a 2 x 3 factorial design to test the effects of plane of nutrition (diet) and stage of gestation on maternal visceral tissue mass, intestinal cellularity, crypt cell proliferation, and jejunal mucosal vascularity. Singleton pregnancies to a single sire were established by embryo transfer, and thereafter ewes were offered a control (Control) or high (High) amount of a complete diet (2.84 Mcal/kg and 15.9% CP; DM basis) to promote slow or rapid maternal growth rates. After d 90 of gestation, feed intake of the Control group was adjusted weekly to maintain BCS and meet the increasing nutrient demands of the gravid uterus. Ewes were slaughtered at 50 d (n = 6 Control; n = 5 High), 90 d (n = 8 Control; n = 6 High), or 130 d (n = 8 Control; n = 6 High) of gestation. Ewes were eviscerated and masses of individual organs were recorded. The jejunum was sampled and processed for subsequent analyses. Final ewe BW for Control-fed ewes was similar at d 50 and 90 and increased (P = 0.10) from d 90 to 130 (46.0, 48.9, and 58.2 +/- 1.6 kg, respectively), whereas final BW increased (Por= 0.01) throughout gestation in High-fed ewes (58.3, 68.8, and 81.1 +/- 1.6 kg, respectively). Relative jejunum mass (g/kg of maternal BW) was greater (P = 0.003) in Control-fed ewes compared with High-fed ewes and tended (P = 0.11) to decrease from d 50 to 130. There were diet x stage of gestation interactions (Por= 0.08) for ileum and small intestinal total and relative weights. Ileum mass (g/kg of maternal BW) in Control-fed ewes was less (P = 0.07) compared with High-fed ewes at d 50, was equal (P = 0.19) to High-fed ewes at d 90, and was greater (P = 0.02) than High-fed ewes at d 130. Small intestine mass (g/kg of maternal BW) was similar between Control- and High-fed ewes at d 50 and 90, but Control-fed ewes had greater (P = 0.01) mass at d 130. Jejunal RNA and protein concentrations were less (Por= 0.07) and DNA was unaffected (P = 0.43) in Control-fed compared with High-fed ewes. Stage of gestation did not affect jejunal RNA, DNA (mg/g), or protein concentrations. Jejunal cellular proliferation was not affected by diet or stage of gestation. In jejunal mucosal tissue, capillary number decreased, whereas capillary surface density and area per capillary increased (P = 0.01) with advancing pregnancy (d 50 vs. d 130), but were independent of diet. Data indicated that intestinal mass as a proportion of maternal BW declined in overnourished, gestating ewe lambs. This response was more pronounced during late gestation and is likely explained by the increasing maternal BW and adiposity rather than by the changing cellularity or cell proliferation.

To examine effects of nutritional plane and Se supplementation on colostrum quality and mammary development, individually fed, pregnant Rambouillet ewe lambs were allotted randomly to 1 of 6 treatments in a 2 x 3 factorial arrangement. Main effects included dietary Se level, which began at breeding (d = 0) [adequate Se (9.5 mug/kg of BW) vs. high Se (81.8 mug/kg of BW)], and plane of nutrition, which began at d 50 of gestation [60% (RES), 100% (CON), and 140% (HIGH) of requirements]. Upon parturition, lambs were immediately separated from dams and weighed. Three hours after lambing, colostrum yield was determined, and samples were obtained for components and immunoglobulin G (IgG) analysis. Ewes were slaughtered within 24 h of parturition, and mammary tissues were collected for determination of alveolar secretory epithelial cell proliferation index and luminal area. Gestation length was reduced (P0.01) in HIGH ewes compared with RES and CON ewes. Although birth weights were reduced (P0.01) in RES and HIGH compared with CON ewes, there was little effect of diet on placental size. Mammary gland weight was reduced (P/= 0.05) in RES compared with CON and HIGH, which were similar. However, when expressed as grams per kilogram of BW, mammary gland weight in HIGH ewes was less (P = 0.03) compared with RES and CON. Colostrum weight and volume were reduced (P0.01) in RES and HIGH ewes compared with CON. Although colostrum IgG concentration was greater in RES ewes compared with CON and HIGH, total IgG was lower (P/= 0.06) in RES and HIGH compared with CON ewes. The percentage of alveolar cells proliferating was increased (P0.04) in HIGH compared with RES ewes, with CON being intermediate. Percentage of alveoli luminal area per unit tissue area was increased (P = 0.04) in RES compared with HIGH and CON ewes, which did not differ. Selenium had no effect (P/= 0.15) on mammary gland weight, colostrum quantity, or IgG concentration in pregnant ewe lambs. Improper nutrition from mid to late pregnancy in ewe lambs altered colostrum quality and quantity and reduced offspring birth weight, which may have negative implications for lamb health and survival during the early postnatal period.

The objective of these studies was to evaluate the effects of dietary restriction and Se on maternal and fetal metabolic hormones. In Exp. 1, pregnant ewe lambs (n = 32; BW = 45.6 +/- 2.3 kg) were allotted randomly to 1 of 4 treatments. Diets contained (DM basis) either no added Se (control), or supranutritional Se added as high-Se wheat at 3.0 mg/kg (Se-wheat), or sodium selenate at 3 (Se3) and 15 (Se15) mg/kg of Se. Diets (DM basis) were similar in CP (15.5%) and ME (2.68 Mcal/kg). Treatments were initiated at 50 +/- 5 d of gestation. The control, Se-wheat, Se3, and Se15 treatments provided 2.5, 75, 75, and 375 microg/kg of BW of Se, respectively. Ewe jugular blood samples were collected at 50, 64, 78, 92, 106, 120, and 134 d of gestation. Fetal serum samples were collected at necropsy on d 134. In Exp. 2, pregnant ewe lambs (n = 36; BW 53.8 +/- 1.3 kg) were allotted randomly to treatments in a 2 x 2 factorial arrangement. Factors were nutrition (control, 100% of requirements vs. restricted nutrition, 60% of control) and dietary Se (adequate Se, 6 microg/kg of BW vs. high Se, 80 microg/kg of BW). Selenium treatments were initiated 21 d before breeding, and nutritional treatments were initiated on d 64 of gestation. Diets were 16% CP and 2.12 Mcal/kg of ME (DM basis). Blood samples were collected from the ewes at 62, 76, 90, 104, 118, 132, and 135 d of gestation. Fetal blood was collected at necropsy on d 135. In Exp.1, dietary Se source and concentration had no effect (P0.17) on maternal and fetal serum IGF-I, triiodothyronine (T(3)), or thyroxine (T(4)) concentrations. Selenium supplementation increased (P = 0.06) the T(4):T(3) ratio vs. controls. In Exp. 2, dietary Se had no impact (P0.33) on main effect means for maternal and fetal serum IGF-I, T(3), or T(4) concentrations from d 62 to 132; however, at d 135, high-Se ewes had lower (P = 0.01) serum T(4) concentrations than adequate-Se ewes. A nutrition by Se interaction (P = 0.06) was detected for the T(4):T(3) ratios; ewes fed restricted and adequate-Se diets had greater (P = 0.10) T(4):T(3) ratios compared with the other treatments. Nutrient-restricted ewes had lower (P0.05) serum IGF-I, T(3), and T(4) concentrations. Fetal serum IGF-I concentrations were lower (P = 0.01) in restricted-vs. control-fed ewes; however, fetal T(3) and T(4) concentrations were unaffected (P0.13) by dietary Se or maternal plane of nutrition. These data indicate that dietary Se may alter maternal T(4):T(3) ratios. In addition, nutrient restriction during gestation reduces maternal IGF-I, T(3), and T(4) and fetal IGF-I concentrations.

Pregnant Targhee ewe lambs (n = 32; BW = 45.6 +/- 2.2 kg) were allotted randomly to 1 of 4 treatments in a completely randomized design to examine the effects of level and source of dietary Se on maternal and fetal visceral organ mass, cellularity estimates, and maternal jejunal crypt cell proliferation and vascularity. Diets contained (DM basis) either no added Se (control) or supranutritional Se from high-Se wheat at 3.0 ppm Se (SW) or from sodium selenate at 3 (S3) or 15 (S15) ppm Se. Diets were similar in CP (15.5%) and ME (2.68 Mcal/kg of DM) and were fed to meet or exceed requirements. Treatments were initiated at 50 +/- 5 d of gestation. The control, SW, S3, and S15 treatment diets provided 2.5, 75, 75, and 375 microg of Se/kg of BW, respectively. On d 134 +/- 10 of gestation, ewes were necropsied, and tissues were harvested. Contrasts, including control vs. Se treatments (SW, S3, and S15), SW vs. S3, and S3 vs. S15, were used to evaluate differences among Se levels and sources. There were no differences in ewe initial and final BW. Full viscera and liver mass (g/kg of empty BW and g/kg of maternal BW) and maternal liver protein concentration (mg/g) and content (g) were greater (P0.04) in Se-treated compared with control ewes. Maternal liver protein concentration was greater (P = 0.01) in SW vs. S3 ewes, and content was greater (P = 0.01) in S15 compared with S3 ewes. Maternal jejunal mucosal DNA concentration (mg/g) was greater (P = 0.08) in SW compared with S3 ewes. Total number of proliferating cells in maternal jejunal mucosa was greater (P = 0.02) in Se-fed compared with control ewes. Capillary number density within maternal jejunal tissue was greater (P = 0.08) in S3 compared with SW ewes. Selenium treatment resulted in reduced fetal heart girth (P = 0.08). Fetal kidney RNA (P = 0.04) and protein concentrations (mg/g; P = 0.03) were greater in Se-treated compared with control ewes. These results indicate that supranutritional dietary Se increases cell numbers in maternal jejunal mucosa through increased crypt cell proliferation. No indications of toxicity were observed in any of the Se treatments.

To determine the relationships among vascularity, expression of angiogenic factors, and selected intrafollicular factors in dominant and nondominant follicles of the first follicular wave, ovaries were obtained on d 3 of the estrous cycle from mature cross-bred beef heifers (n = 8) after a synchronized estrus. Follicular fluid (FF) was collected from all folliclesor = 3 mm for determination of estradiol-17beta (E), progesterone (P4), vascular endothelial growth factor (VEGF), and IGFBP concentrations. The ovaries were then perfusion-fixed and used for histochemical detection of lectin BS-1 (a marker of endothelial cells and thus vascularization) binding, and immunolocalization of VEGF, endothelial nitric oxide synthase (eNOS), and proliferating cell nuclear antigen, followed by image analysis of selected follicles. Follicles were classified, based on E and P4 concentrations in FF, as dominant, estrogen-active (EA; E:P4or = 1) or nondominant, estrogen-inactive (EI; E:P41). Concentrations of E and VEGF in FF, the area of positive staining for lectin BS-1, VEGF, and eNOS, and the labeling index (an index of the percentage of cells proliferating) in granulosa and theca layers were greater (P0.05) in the EA than in the EI follicles, but concentrations of P4 and IGFBP in FF were less (P0.05) in EA than in EI follicles. In addition, vascularity was positively correlated (P0.05) with VEGF and eNOS protein expression, and tended (P0.1) to be positively correlated with the E:P4 ratio in FF but tended (P0.1) to be negatively correlated with IGFBP and P4 concentrations in FF. These data highlight the importance of vascularity, angiogenic factors, and IGFBP in the health of the dominant follicle in heifers, and indicate that the FF concentrations of E, VEGF, IGFBP, and P4, and the E:P4 ratio can be used as markers of dominant follicles.

Soon after ovulation, the corpus luteum (CL) starts secreting progesterone (P(4)), a hormone necessary for implantation. The aim of the study was to evaluate whether P(4) exerts an autocrine/paracrine action on luteal angiogenic activity and P(4), prostaglandin E(2) (PGE(2)) and NO production in the mare. Corpora hemorrhagica (CH) and mid-luteal phase CL (MCL) were cultured with (i) no hormone (Control); (ii) P(4); (iii) a P(4) precursor - pregnenolone; or (iv) a P(4) antagonist - onapristone [10(-4) M;10(-5) M; all steroids]. NO production decreased in MCL, with respect to CH, when treated with P(4) [10(-4) M] and pregnenolone [10(-5) M]. PGE(2) increased from CH to MCL, and showed a tendency to rise in pregnenolone treated luteal tissues (10(-4) M; p=0.06). In the CH, P(4) decreased with pregnenolone [10(-4) M] compared to control, P(4) [10(-5) M], onapristone [10(-4) M;10(-5) M] and pregnenolone [10(-5) M](p0.05). In the MCL, pregnenolone [10(-5) M] decreased (p0.05) and P(4) tended to decrease (p=0.06) bovine aortic endothelial cell (BAEC) mitogenesis. Onapristone [10(-4) M] increased BAEC proliferation with respect to P(4) (p=0.01). Since there was no direct effect of treatments on BAEC, these data suggest that long-lasting effects of P(4) and its precursor may inhibit angiogenic factor(s) production by equine MCL, preparing for CL functional and structural regression.

Nutrition has been shown to influence several reproductive functions, including hormone production, oocyte competence and fertilization, and early embryonic development. To determine the effects of maternal diet on in vitro fertilization (IVF) and early embryonic development, ewes (n = 18; 47.0 +/- 1.5 kg of initial BW) were divided into control and underfed (60% of control) nutritional planes for 8 wk before oocyte collection. Pelleted diets containing 2.4 Mcal of ME/kg and 13% CP (DM basis) were fed once daily. During the first 4-wk acclimation phase, control and underfed ewes were fed 1,000 and 600 g/d, respectively. From wk 4 to 8, control (adequate) ewes were fed to maintain BW and offered 720 g/d, whereas underfed ewes received 432 g/d (60% restricted). Synchronization of estrus was performed using progestagen sponges for 14 d. Follicular development was induced by twice daily injections of FSH on d 13 (5 units/injection) and 14 (4 units/injection) of the estrous cycle. Oocytes were collected from all visible follicles on d 15 of the estrous cycle. After IVF, the proportion of developing embryos was evaluated throughout an 8-d culture period. Under-nutrition decreased (P0.006) the rate of cleavage, number of blastocysts per ewe, and rate of blastocyst formation (from 79 to 64%; from 3.3 to 0.8; and from 31 to 8%, respectively). However, the number of visible follicles, total number of oocytes, number of healthy oocytes, percentage of healthy oocytes, number of cleaved oocytes, and morula formation per ewe were similar for control and underfed ewes. These data indicate that undernutrition of donor ewes, resulting in lower BW and BCS, has a negative effect on oocyte quality, which results in lower rates of cleavage and blastocyst formation.

The objectives of this study were to evaluate intestinal cellularity and vascularity in mature ewes in response to dietary restriction and pregnancy status and to quantify the response of these variables to increased nutrient demand of fetal growth. In Exp. 1, 28 mature Dorset x crossbred white-faced ewes (61.6+/-1.8 kg initial BW) were fed a pelleted, forage-based diet. Treatments were arranged in a 2 x 3 factorial, with dietary restriction (60% restriction vs. 100% maintenance for respective states of pregnancy) and pregnancy status (nonpregnant, NP; d 90 and 130) as main effects. Dietary treatments were initiated on d 50 of gestation and remained at 60 or 100% maintenance throughout the experiment. Nonpregnant ewes were fed dietary treatments for 40 d. In Exp. 2, four Romanov ewes were naturally serviced (Romanov fetus and Romanov dam; R/R); two Romanov embryos per recipient were transferred to four Columbia recipients (Romanov fetus and Columbia recipient; R/C), and three Columbia ewes were naturally serviced (Columbia fetus and Columbia dam; C/C). In Exp. 1, dietary restriction and pregnancy status interacted with regard to maternal jejunal DNA concentration (P0.01), with restricted ewes having a greater DNA concentration (mg/g; fresh basis) at d 130. Vascularity (percentage of total tissue area) in the jejunum was increased (P0.06) as a result of dietary restriction and pregnancy status. Total microvascular volume ofjejunal tissue was not altered by dietary restriction and increased (P0.01) at d 130 of pregnancy. In Exp. 2, R/R ewes had less (P0.09) DNA (g) in the jejunum compared with R/C and C/C ewes. Jejunal vascularity (%) was increased (P0.05) in R/R ewes compared with R/C or C/C ewes, whereas total jejunal microvascular volume remained unchanged. These data demonstrate intestinal vascular density responds to changes in diet and physiological state. In addition, pregnancy increased total jejunal microvascular volume.

Our objectives were to evaluate maternal body changes in response to dietary restriction or the increased nutrient requirement of fetal growth. In Exp. 1, 28 mature crossbred ewes (61.6 +/- 1.8 kg initial BW) were fed a pelleted forage-based diet to evaluate effects of pregnancy and nutrient restriction on visceral organ mass. Treatments were arranged in 2 x 3 factorially, with dietary restriction (60% restriction vs. 100% maintenance) and reproductive status (nonpregnant [NP], d 90 or d 130 of gestation) as main effects. Dietary treatments were begun at d 50 of gestation, and restricted ewes remained at 60% of maintenance throughout the experiment. Nonpregnant and d-90 ewes were fed dietary treatments for 40 d and slaughtered. The d-130 ewes were fed dietary treatments for 80 d and then slaughtered. In Exp. 2, four Romanov ewes were naturally mated (Romanov fetus and Romanov dam; R/ R), and two Romanov embryos were transferred to each of four Columbia recipients (Romanov embryos and Columbia recipient; R/C). Three Columbia ewes were naturally mated (Columbia fetus and Columbia recipient; C/C). In both experiments, maternal organ weights were reported as fresh weight (grams), scaled to empty body weight (EBW; grams per kilogram) and maternal body weight (MBW; grams per kilogram). In Exp. 1, ewe EBW and fetal mass were decreased (P0.02) with restriction compared with maintenance. Dietary restriction decreased liver mass (16.7 vs. 14.5 g/kg EBW or 18.8 vs. 16.4 g/kg MBW; P0.01), but dietary restriction did not affect total digestive tract mass. In Exp. 2, ewe BW was less for the R/R compared with R/C and C/C (44.8 vs. 110.4 and 98.1 +/- 7.9 kg, respectively; P0.01). Fetal weight at d 130 was less for the R/R than for R/C and C/C (2.2 vs. 3.3 and 4.7 +/- 0.3 kg, respectively; P0.01) when measured as individual fetuses; however, when measured as total fetal mass carried in each ewe, there was no effect of ewe type. These data suggest that the gastrointestinal tract, along with other maternal organs, responds to both level of dietary intake and nutrient requirements for gestation, and that fetal weight is decreased as a result of a 40% decrease in nutrients offered.

Beef heifers (24 mo; 378 +/- 32 kg of BW; 22 pregnant, PR; 17 nonpregnant, NP) were grouped in common pens and fed corn silage- and hay-based diets formulated to provide an ADG of 0.45 kg in NP heifers. Both PR and NP heifers were slaughtered on d 40, 120, 200, and 270 of the study. Intestinal and hepatic tissues were analyzed for protein, DNA, RNA (mg/g of fresh tissue), and in vitro oxygen use. Jejunal samples were analyzed for cellular proliferation via immunohistochemical analysis. For ileum, DNA, which provides an estimate of cell number per unit of tissue, revealed an interaction (P = 0.06) between pregnancy and slaughter day; both PR and NP decreased with time, but NP increased on d 270 (P = 0.09). Cell number in the ileum was reduced at d 200 and 270 in the PR heifers (P0.09). Liver protein concentration was less (P = 0.07) in PR than in NP heifers (NP = 291.1 vs. PR = 210.5 +/- 33.9 mg/g). Hepatic protein:DNA ratio was not affected (P0.10) by pregnancy or day. Energy use (kcal/d) of duodenum and jejunum, calculated from in vitro oxygen consumption, increased linearly (P0.02) with time for both PR and NP. Pregnant and NP ileal energy use increased linearly (P0.01), but ileal energy use by PR was less throughout gestation (P = 0.07) than ileal energy use by NP. Cellular proliferation in the crypt region of the jejunum was decreased on d 120 and 200 (P0.02). These data indicate that the small intestine and liver of PR heifers may conserve energy expenditure compared with NP heifers. Energy conservation can partially be explained by differences in growth and cell proliferation and by energy use of the liver and small intestine.

Corpora lutea (CL) from Days 5, 10, and 15 after superovulation were enzymatically dispersed, and a portion of the cells were elutriated to obtain fractions enriched with small or large luteal cells. Mixed, small, and large luteal cell fractions were incubated with no treatment or with agonists or antagonists of cAMP (dbcAMP or Rp-cAMPS), protein kinase C (PKC; TPA or H-7), or calcium (A23187, EGTA, or A23187 + EGTA). The rate of contact-dependent gap junctional intercellular communication (GJIC) was evaluated by laser cytometry. Media were collected for progesterone (P(4)) radioimmunoassay, and luteal cells cultured with no treatment were fixed for immunocytochemistry or frozen for Western blot analysis. Luteal cells from each stage of the estrous cycle exhibited GJIC. The dbcAMP increased (P0.05) GJIC for all cell types across the estrous cycle. The Rp-cAMPS decreased (P0.05) GJIC for small luteal cells on Day 5 and for all cell types on Days 10 and 15. The TPA inhibited (P0.01), but H-7 did not affect, GJIC for all cell types across the estrous cycle. The A23187 decreased (P0.05) GJIC for large luteal cells touching only small or only large luteal cells, whereas A23187 + EGTA decreased (P0.05) GJIC for all cell types across the estrous cycle. For the mixed and large luteal cell fractions, dbcAMP increased (P0.05), but TPA and A23187 + EGTA decreased (P0.05), P(4) secretion. The A23187 alone decreased (P0.05) P(4) secretion by large, but not by mixed, luteal cells. For all days and cell types, the rate of GJIC and P(4) secretion were correlated (r = 0.113-0.249; P0.01). Connexin 43 was detected in cultured luteal cells by immunofluorescence and Western immunoblotting. Thus, intracellular regulators like cAMP, PKC, or calcium appear to regulate GJIC, which probably is an important mechanism for coordinating function of the ovine CL.

Because of rapid growth followed by spontaneous regression, the ovarian corpus luteum (CL) is an excellent model to study angiogenesis in vivo. To evaluate the expression of vascular endothelial growth factor (VEGF) protein during luteal development, ovaries were collected from FSH-stimulated ewes throughout the estrous cycle. VEGF was immunolocalized in tissue sections by using an affinity-purified antibody. VEGF protein localized exclusively to the thecal layer of preovulatory follicles, while the granulosa was devoid of staining. Associated with the periovulatory period was intense expression of VEGF by thecal cells at the basement membrane and subsequent invasion of the granulosa layers by these VEGF-positive cells immediately after ovulation. The early CL showed staining for VEGF in thecal-derived compartments, and strong staining for VEGF was also seen in cells within the granulosa-derived parenchymal lobules. Dual immunohistochemical localization of VEGF and smooth muscle cell alpha-actin indicated that the VEGF-positive cells were capillary pericytes or vascular smooth muscle cells. In another experiment, we quantified proliferation of endothelial cells and pericytes throughout luteal development. Pericytes represented a large proportion of the proliferating cells during the early luteal phase and then decreased dramatically. Perivascular cells, therefore, may play a critical role in angiogenesis that occurs during transformation of the follicle into the highly vascular CL of the sheep. As angiogenesis occurs only at the level of capillaries, and pericytes are integral members of these microvessels, regulation of pericytes may provide a novel mechanism for regulating luteal growth and tissue growth in general.

The mammalian placenta is the organ through which respiratory gases, nutrients, and wastes are exchanged between the maternal and fetal systems. Thus, transplacental exchange provides for all the metabolic demands of fetal growth and development. The rate of transplacental exchange depends primarily on the rates of uterine (maternal placental) and umbilical (fetal placental) blood flows. In fact, increased uterine vascular resistance and reduced uterine blood flow can be used as predictors of high risk pregnancies and are associated with fetal growth retardation. The rates of placental blood flow, in turn, are dependent on placental vascularization, and placental angiogenesis is therefore critical for the successful development of viable, healthy offspring. Recent studies, including gene knockouts in mice, indicate that the vascular endothelial growth factors represent a major class of placental angiogenic factors. Other angiogenic factors, such as the fibroblast growth factors or perhaps the angiopoietins, also may play important roles in placental vascularization. In addition, recent observations suggest that these angiogenic factors interact with the local vasodilator nitric oxide to coordinate placental angiogenesis and blood flow. In the future, regulators of angiogenesis that are currently being developed may provide novel and powerful methods to ensure positive outcomes for most pregnancies.

Uterine expression of angiogenic factors (vascular endothelial growth factor [VEGF] and basic fibroblast growth factor [bFGF]) was evaluated in ovariectomized ewes at 0, 2, 4, 8, 24, 48, or 72 h after estradiol (E2) treatment. Endometrial VEGF mRNA increased more than 5-fold from 0 to 4 h, remained elevated at 8 h, and then declined through 72 h after E2 treatment. In contrast, endometrial bFGF mRNA remained constant from 0 to 4 h, increased 2.2-fold from 4 to 8 h, remained elevated at 24 h, and then declined through 72 h. Immunostaining for VEGF was present in myometrial and endometrial microvessels (arterioles, venules, and/or capillaries) and also in myometrial smooth muscle; the pattern of VEGF immunostaining followed that of mRNA expression, being elevated at 4 and 8 h after E2 treatment. Immunostaining for bFGF was present exclusively in uterine glands; the pattern of bFGF immunostaining also followed that of its mRNA, being elevated at 8 and 24 h after E2. On the basis of these observations, we suggest that VEGF and bFGF are probably important factors responsible for the dramatic uterine microvascular response that occurs 8 to 24 h after E2 treatment in ovariectomized ewes.

The time-course of uterine growth, cell proliferation, and microvascular development was evaluated during the first 72 h after implanting estradiol-17beta (E2) into ovariectomized (OVX) ewes. Uterine fresh weight increased 2.3-fold by 24 h and increased further (3.3-fold) by 48 h. The majority (approximately 75%) of this growth response was associated with tissue growth rather than a change in the tissue dry weight:fresh weight ratio. Both uterine cell number (DNA content) and cell size (RNA:DNA ratio) increased from 0 to 24 h (1.8-fold and 1.7-fold, respectively). Cell proliferation also increased dramatically between 8 h and 24 h after E2 implantation. Endometrial microvascular volume density (percentage of tissue volume occupied by microvessels) increased approximately 1.8-fold by 24 h and then remained constant or declined slightly through 72 h. The total endometrial microvascular volume, however, increased approximately 5-fold from 0 to 24 h and increased further by 72 h. Thus, treatment of OVX ewes with E2 caused a dramatic increase in uterine fresh and dry weights by 24 h, due primarily to hyperplasia and hypertrophy, with only a relatively small change in tissue dry weight:fresh weight ratio. This dramatic uterine growth was associated with a profound increase in endometrial microvascular volume.

Uterine growth, morphology, and endometrial cell proliferation were evaluated in intact estrous (EST; Day 0 of the estrous cycle) or luteal (LUT; Day 10 of the estrous cycle) ewes or in ovariectomized ewes receiving no hormone (OVX); estradiol-17beta (OVX+E); progesterone for 8 (OVX+P8), 30 (OVX+P30), or 60 (OVX+P60) days; estradiol followed by progesterone (OVX+E+P); or progesterone followed by estradiol (OVX +P+E). Uterine weights were greater for EST than for LUT ewes, were reduced 2- to 4-fold in OVX ewes, and were increased in estradiol-treated (OVX+E, OVX+E+P, and OVX+P+E) ewes. The effects of estradiol treatment on uterine growth in OVX ewes were primarily on cellular hypertrophy rather than hyperplasia. Endometrial labeling index (LI) was greater for EST than for LUT ewes and was low in OVX ewes. Compared with OVX ewes, OVX+E and OVX+P+E ewes had increased LI for all endometrial tissues, whereas OVX+P8 and OVX+E+P ewes had increased LI only for luminal epithelium. These data will help us determine the mechanisms by which ovarian steroids regulate uterine growth in ewes.

Uterine growth, cell proliferation, and endometrial expression of the c-fos proto-oncogene were evaluated for nonpregnant ewes (n = 6 ewes per day) on Days 0 (estrus), 2, 4, 8, 12, and 15 of the estrous cycle. Fresh and dry weights of uterine horns decreased (p0.01) linearly from Days 0 to 8 and then remained similar from Day 8 through Day 15. Tissue water content (1 - [dry weight/fresh weight]) was greater on Day 0 (p0.01) than on Days 2, 4, and 8, and the latter values were greater (p0.01) than on Days 12 and 15. DNA content was similar on Days 0, 2, 4, 8, and 15 but increased (p0.01) on Day 12. Although DNA content was greatest on Day 12, the ratios of RNA to DNA and of protein to DNA were least (p0.01) on that day. Thus, changes in uterine fresh weight were associated primarily with changes in tissue hypertrophy (RNA:DNA and protein:DNA ratios) and water content. In addition, changes in uterine fresh weight were associated with changes in the ratio of estradiol to progesterone in systemic blood, which was greatest (p0.01) on Days 0 and 2, decreased (p0.01) from Day 2 to Day 8, remained low through Day 12, and then was elevated again (p0.01) on Day 15. Moreover, compartmentalized changes in endometrial cell proliferation (labeling index [LI]; percentage of cells exhibiting nuclear incorporation of bromodeoxyuridine, a thymidine analogue) also were associated with the changes in fresh weight. The epithelium of the uterine lumen and luminal glands exhibited the greatest changes in rate of cell proliferation and accounted for most of the changes in LI seen across days of the estrous cycle. Endometrial expression of c-fos mRNA and protein also reflected changes in uterine weight and the systemic estradiol:progesterone ratio. The level of c-fos mRNA was greatest at estrus, low on Days 2-8, and elevated slightly on Days 12 and 15; Fos protein was greatest on Days 0 through 4, least on Day 8, and intermediate on Days 12 and 15. Characterization of uterine growth, cell proliferation, and c-fos expression throughout the estrous cycle will provide a foundation for future studies of gene expression regulating growth of the nonpregnant uterus.

The corpus luteum undergoes tremendous growth, development and regression each oestrous or menstrual cycle. These changes are reflected by equally impressive growth and regression of the luteal vasculature. We have previously shown that angiogenic factors from corpora lutea are primarily heparin binding and that one of these factors is similar to vascular endothelial growth factor (VEGF). In an effort to identify this factor, and to define its role in luteal vascular development, the cDNA for the coding region of ovine VEGF was sequenced and a sensitive RNase protection assay was developed to quantitate mRNA encoding VEGF in luteal tissues from ewes in the early (days 2\p=n-\4),mid- (day 8) and late (days 14\p=n-\15)stages of the oestrous cycle. In addition, an N-terminal peptide was synthesized from the translated ovine cDNA sequence for VEGF and an antiserum was raised against this peptide for use in western immunoblotting procedures. Nested reverse transcriptase (RT)-PCR of RNA from ovine corpora lutea resulted in three products that correspond in size to the alternatively spliced variants of VEGF VEGF120, VEGF164, and VEGF188) predicted from other species. The RNase protection assay revealed that the proportion of mRNA encoding VEGF was 2- to 3-fold greater on days 2\p=n-\4than on day 8 or days 14\p=n-\15.Densitometric analysis of gels from the RNase protection assay showed that VEGF120 represented approximately one third of the total mRNA encoding VEGF in the corpus luteum and that this proportion did not vary with stage of the oestrous cycle. SDS-PAGE and western immunoblot analysis of a homogenate from corpora lutea showed a single 18 kDa protein. These data demonstrate that VEGF is expressed in luteal tissue throughout the ovine oestrous cycle and that expression of mRNA encoding VEGF is upregulated during the period of rapid luteal development, when luteal vascular growth is at its maximum.

To determine the effects of FSH-P treatment and subsequent withdrawal on follicular growth, cell proliferation, and atresia, ewes (n = 4 ewes/treatment group) received twice daily injections of saline or FSH-P beginning on Day 13 of the estrous cycle (length of the estrous cycle = 16.5 days) and were slaughtered after 0, 48, or 72 h of treatment (i.e., on Days 13, 15, or 16). Some treatment groups received FSH-P from Day 13 until slaughter (FSH-P-treated), whereas some received FSH-P for 24-48 h followed by saline for 24-48 h (FSH-P withdrawal). All ewes received an i.v. injection of bromodeoxyuridine (BrdU, a thymidine analogue) 1 h before slaughter. For both ovaries from each ewe, the number and surface diameter of all visible antral follicles were determined, and antral follicles were classified as small (or = 3 mm), medium (3 mm toor = 6 mm), or large (6 mm). As an index of the rate of cell proliferation, BrdU was immunolocalized in paraffin-embedded tissue sections, and the labeling index (LI; BrdU-labeled nuclei as a percentage of total nuclei) was determined for granulosa and thecal cells of nonatretic and early atretic antral follicles of known diameter. Follicular status (atretic vs. nonatretic) was evaluated morphologically. Moreover, the presence of apoptosis was detected in situ by using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling method. For untreated and saline-treated ewes, the number of small follicles per ewe increased (p0.01) from Day 13 to Day 15, then decreased again on Day 16, whereas numbers of medium and large follicles did not differ across days. Compared with saline-treated ewes, ewes receiving FSH-P from Day 13 until slaughter had fewer (p0.05) small but more (p0.05) medium and large follicles. Compared with FSH-P-treated ewes, FSH-P withdrawal resulted in fewer (p0.05) medium and large but more (p0.05) small follicles. Across all follicular size classes, granulosa and thecal cell LI of nonatretic follicles was decreased (p0.05) by FSH-P withdrawal compared with FSH-P treatment. Additionally, across all follicular size classes, FSH-P withdrawal increased (p0.01) the percentage of follicles that were atretic compared with saline or FSH-P treatment. Histochemical staining of early and advanced atretic follicles showed that granulosa cells are the predominant site of cell death (apoptosis) during follicular atresia. Thus, compared with continuous FSH-P treatment, withdrawal of FSH-P resulted in decreased numbers of medium and large follicles, decreased proliferation of follicular cells, and an increased incidence of atresia associated with granulosa cell death. This model should prove useful for studying the mechanisms regulating follicular growth and atresia in ewes.

The present study examined the pattern of expression of the gap junctional protein connexin 43 (Cx43) in bovine corpora lutea (CL) during growth, differentiation, and regression. CL from the early (n = 6), mid- (n = 6), and late (n = 6) luteal phases of the estrous cycle were weighed and divided into several portions. One portion of each CL was frozen in liquid nitrogen for evaluation of protein, DNA, progesterone, and presence of Cx43 by Western immunoblot analysis; another portion was frozen in liquid propane for immunofluorescent staining of Cx43. An additional portion of each CL was dispersed, and the luteal cells were cultured for 2 days, fixed, and used for immunofluorescent staining of Cx43. Weights and DNA, protein, and progesterone contents of CL increased (p0.05) from the early to mid-luteal phases and then decreased (p0.05) from the mid- to late luteal phases. The ratio of protein to DNA was similar in the early and mid-luteal phases and then decreased (p0.05) to the late luteal phase. Western immunoblot analysis revealed bands at 43 kDa that differed in volume (evaluated by densitometry); the early luteal phase volume was greater (p0.05) than that at the mid-luteal phase, which was greater (p0.05) than that at the late luteal phase. Immunofluorescent staining demonstrated that Cx43 was present in luteal tissues and cultured luteal cells throughout the estrous cycle, and the area of positive staining decreased (p0.05) as the estrous cycle progressed. Staining for Cx43 was punctate and localized to the cellular borders. Thus, levels of Cx43 in bovine CL are greatest early in the estrous cycle and are least late in the estrous cycle. These data demonstrate that gap junctions may be important for regulation of luteal growth, differentiation, and regression in the cow.

Cellular interactions mediated by both contact-dependent and contact-independent mechanisms are probably important to maintain luteal function. The objective of the present study was to evaluate the role of cAMP in regulation of contact-dependent gap junctional intercellular communication (GJIC) of bovine luteal cells from several stages of luteal development. In experiment 1, corpora lutea (n = 5) from the mid-luteal phase of the estrous cycle were dissociated with collagenase, and cells were preincubated in a medium with serum. Then the medium was changed to serum-free media containing a cAMP agonist (dbcAMP; 1 mM) or antagonist (Rp-cAMPS; 0, 3, 10, 30, or 100 microM). In experiment 2, corpora lutea from the early (n = 7), mid- (n = 6), and late (n = 6) luteal phases of the estrous cycle were dissociated and preincubated as in experiment 1, and luteal cells were then incubated with no treatment, LH (100 ng/ml), dbcAMP (1 mM), forskolin (1 microM), Rp-cAMPS (100 microM), or LH+Rp-cAMPS. After incubation of luteal cells with treatments for 18-24 h, media were collected for determination of progesterone and cAMP concentrations. Then the rate of GJIC was evaluated for selected cells (small luteal cells in contact with small luteal cells, and large luteal cells in contact with small luteal cells) by using the fluorescence recovery after photo-bleaching technique and laser cytometry. In experiment 1, dbcAMP increased (p0.01) but Rp-cAMPS (p0.05) decreased GJIC between small luteal cells and between large and small luteal cells. In addition, dbcAMP stimulated (p0.01) but Rp-cAMPS did not affect progesterone secretion. In experiment 2, treatments affected (p0.05) GJIC and progesterone production of luteal cells from the mid- and late but not from the early luteal phase of the estrous cycle. GJIC between small luteal cells was increased (p0.01) by LH, dbcAMP, and forskolin. GJIC between large and small luteal cells was increased (p0.05) by dbcAMP and forskolin. Rp-cAMPS decreased (p0.01) GJIC between small luteal cells (mid-luteal phase) and between large and small luteal cells (mid- and late luteal phases). In addition, Rp-cAMPS inhibited (p0.05) the stimulatory effects of LH on GJIC between small luteal cells from the mid- and late luteal phases of the estrous cycle. For luteal cells from the mid- and late luteal phases, progesterone production was increased (p0.05) by LH, dbcAMP, forskolin, and LH+Rp-cAMPS, but was not affected by Rp-cAMPS. Across all stages of the estrous cycle, cyclic AMP accumulation in media was greater (p0.05) in LH- and forskolin-treated cultures than in control cultures and was greater (p0.01) in forskolin-treated than in LH-treated cultures. These data demonstrate that small and large luteal cells communicate with each other and that the rate of GJIC is modulated by LH and cAMP, as has been shown previously for other cell types. Thus, cAMP appears to be involved in the regulation of GJIC within the bovine corpus luteum, which probably is an important mechanism for coordinating luteal cell function.

Follicular and luteal morphology and steroidogenic function were investigated by immunohistochemistry for cytochrome P450 17 alpha-hydroxylase (P450c17) and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) during the estrous cycle in pigs, sheep, and cows. The theca interna of all species expressed P450c17 during follicular development. In the pig, this constituted a continuous layer of cells around the follicle, but a sheath of cells lining the basement membrane appeared not to express P450c17. Neither was expression of P450c17 in ovine and bovine follicles uniform throughout the theca interna. In these two species, a beaded appearance was given by P450c17, since it was expressed in some regions but not in others. Therefore, staining for P450c17 defined functional sub-populations of cells within the theca interna of pigs, sheep, and cows. Ovulation was associated with a decrease in P450c17 in all species, but some expression persisted in theca-derived cells of developing and mature porcine CL. Expression of 3 beta-HSD in the preovulatory follicle was confined to the theca of the pig and sheep; in contrast, in the cow, it was highest in the granulosa. In general, 3 beta-HSD expression appeared to be greater in porcine than ovine or bovine follicles, the physiological relevance of which is discussed. Porcine and ovine theca continued to express 3 beta-HSD after ovulation, and granulosa-derived cells increased their 3 beta-HSD expression markedly as they luteinized in all three species. During early luteal development in pigs and sheep, theca-derived cells with high 3 beta-HSD encircled luteal lobules, but these cells appeared throughout the parenchyma of the mature CL. Luteal regression in sheep and cows was typified by the loss of many cells expressing 3 beta-HSD, whereas others, adjacent to them, appeared to be intact without loss of enzyme expression. These data further define differences in steroidogenesis during follicular and luteal development among the pig, sheep, and cow.

To evaluate the kinetics of luteal growth, bovine CL were obtained from four stages (stage I, Days 1-4; stage II, Days 5-10; stage III, Days 11-17; stage IV, Days 18-21) of the estrous cycle, and luteal fresh weight as well as DNA, protein, and progesterone contents was determined. To evaluate the relative rate of cell proliferation, proliferating cell nuclear antigen (PCNA; a specific marker for cell proliferation) was immunolocalized in paraffin-embedded luteal tissue sections. To evaluate the relative rate of cell death, nucleosomal fragmentation of DNA (a specific marker for apoptosis) was detected by agarose gel electrophoresis and also by histochemical localization in paraffin-embedded luteal tissue sections. Luteal fresh weight and DNA, protein, and progesterone contents increased (p0.01) from stage I to stage II, were similar between stages II and III, and then decreased (p0.01) from stage III to stage IV. The ratio of protein to DNA (an index of average cell size) was similar for stages I, II, and III and then decreased (p0.01) at stage IV. For stage I (corpora hemorrhagica), most proliferating (PCNA-positive) cells were located in or around the core of the tissue infoldings (presumably thecal-derived areas), whereas relatively few proliferating cells were located at the periphery of the tissue infoldings (presumably granulosa-derived areas). For stages II, III, and IV, the majority of proliferating cells appeared to be small cells (i.e., small parenchymal cells, fibroblasts, and endothelial cells). The labeling index (LI; percentage of cells that were PCNA-positive) was greatest at stage I (20.3 +/- 1.1%); it then decreased (p0.01) by stage II and was similar at stages II, III, and IV (3.4 +/- 1.1%). Apoptosis, as determined by evaluation of nucleosomal DNA fragmentation by agarose gel electrophoresis and in situ localization, was detectable only at stage IV. These data demonstrate that luteal growth from stage I to stage II resulted from cell proliferation as shown by a high LI at stage I, accompanied by increased luteal DNA content but no change in average cell size, and by similar protein: DNA ratios. Luteal regression from stage III to stage IV was primarily associated with cell deletion and decreased cell size as shown by a decrease in luteal DNA content and the appearance of apoptosis along with a decrease in the luteal protein: DNA ratio.(ABSTRACT TRUNCATED AT 400 WORDS)

To determine the effect of exogenous gonadotropin on size, number, cellular proliferation, and atresia of follicles, ewes (n = 3-5/treatment/day) received an injection of vehicle or FSH-P (a pituitary extract) twice daily on Days 13, 14, and 15 (5, 4, and 3 mg FSH-P/injection, respectively; Day 0 = estrus) and were slaughtered on Days 14, 15, or 16 (i.e., after 24, 48, or 72 h of FSH-P treatment, respectively). An additional group of ewes (Day 13 control) received no treatment and were slaughtered on Day 13. All ewes received an i.v. injection of bromodeoxyuridine (BrdU, a thymidine analog; 5 mg/kg-1 BW) 1 h before slaughter. For both ovaries from each ewe, number and surface diameter of all visible follicles were recorded, and antral follicles were classified as small (or = 3 mm), medium (3 mm toor = 6 mm), or large (6 mm). To evaluate rate of proliferation of follicular cells, ovaries were fixed by perfusion with Carnoy's solution, and BrdU was immunolocalized in paraffin-embedded sections by use of a specific primary antibody and indirect immunoperoxidase detection. As an index of the rate of cellular proliferation, labeling index (LI: number of BrdU-labeled nuclei expressed as a percentage of total nuclei) of granulosa and thecal cells was determined by image analysis of antral follicles of known diameter. Follicular status (atretic vs. nonatretic) also was evaluated morphologically by using the histological sections. After 24 h of treatment (i.e., on Day 14), FSH-P-treated ewes had an increased (p0.01) number of medium follicles compared with vehicle-treated ewes.(ABSTRACT TRUNCATED AT 250 WORDS)

Growing pigs (initial BW 14.3 +/- 1.2 kg) were fed isocaloric (3.26 Mcal of ME/kg) and isonitrogenous (16% CP) diets containing either 0 (low fiber, LF; n = 4) or 10% (high fiber, HF; n = 4) wheat straw for ad libitum intake for 14 d. On d 14, each pig was injected i.v. with bromodeoxyuridine (BrdU, a thymidine analog; 5 mg/kg) and was slaughtered 1 h later. Visceral organs (liver, pancreas, and intestines) were weighed, and tissue samples were obtained. Feed consumption, daily gain, gain: feed, and final BW did not differ between treatments. Neither visceral weights nor visceral weights per unit of eviscerated BW were affected by diets. Tissue concentrations of DNA (milligrams/gram of tissue) were lower (P.03) in HF than in LF only for jejunum, ileum, and liver. Contents of DNA and protein (milligrams) did not differ between LF and HF for intestinal segments or liver. Content of RNA (milligrams) was greater (P.04) in HF than in LF only for colon. The number of crypt cell nuclei that were labeled with BrdU (indicating DNA synthesis and thus cell proliferation) was increased (P.03) in HF relative to LF for jejunum and colon. The number of epithelial cells exhibiting DNA fragmentation (indicating programmed cell death) was greater (P.07) in the HF than in the LF group for jejunum and ileum. Width of intestinal villi was increased (P.10) in HF vs LF for jejunum and ileum. Depth of intestinal crypts was increased (P.08) in HF vs LF for jejunum, ileum, and colon.(ABSTRACT TRUNCATED AT 250 WORDS)

Growth and microvascular development of the uterus were evaluated for ewes on Days 12, 18, 24, and 30 after mating (3-4 ewes/day; Day 0 = day of mating) in two experiments. In experiment 1, fresh weight and dry weight of gravid uterine horns were increased on Days 24 and 30 after mating, whereas those of nongravid uterine horns were elevated only on Day 30. The increased fresh and dry weights of gravid uterine horns on Day 24 were associated with uterine hyperplasia (increased DNA content). Increased fresh and dry weights of gravid uterine horns on Day 30, however, were associated with hypertrophy (increased RNA:DNA and protein:DNA ratios) of uterine tissues. In experiment 2, vascularity of endometrial tissues was elevated on Days 24 and 30 after mating. In addition, dramatic changes in uterine architecture (increased lumenal diameter and decreased endometrial thickness) and in uterine microvascular development (increased abundance of large microvessels and development of a subepithelial capillary plexus) were observed by Day 24 after mating. Characterization of the patterns of uterine growth and microvascular development will enable us to further define the role of previously reported uterine and conceptus-derived growth and angiogenic factors during early pregnancy.

In adult tissues, capillary growth (angiogenesis) occurs normally during tissue repair, such as in healing of wounds and fractures. Rampant capillary growth is associated with various pathological conditions, including tumor growth, retinopathies, hemangiomas, fibroses and rheumatoid arthritis. The female reproductive organs (i.e., ovary, uterus, and placenta) exhibit dynamic, periodic growth and regression accompanied by equally dramatic changes in rates of blood flow. It is not surprising, therefore, that they are some of the few adult tissues in which angiogenesis occurs as a normal process. Thus, the female reproductive system provides a unique model for studying regulation of angiogenesis during growth and differentiation of normal adult tissues. Ovarian, uterine, and placental tissues recently have been shown to contain and produce angiogenic and anti-angiogenic factors. This review discusses the current state of knowledge regarding angiogenic processes and their regulation in female reproductive tissues. In addition, implications of this research for regulation of fertility as well as for control of angiogenesis in other normal and pathological processes are discussed.

In the first experiment, minced luteal tissues from cyclic ewes (n = 5) were incubated for 6 h. Media conditioned by these luteal tissue explants stimulated proliferation and migration of endothelial cells. In a second experiment, corpora lutea (CL) from superovulated ewes (n = 12) were dissociated (two ewes/dispersion) and separated into three fractions: a non-elutriated fraction containing a mixed population of luteal cells, a fraction enriched with small steroidogenic luteal cells, and a fraction containing primarily large steroidogenic luteal cells. Fractions (2 X 10(5) viable steroidogenic luteal cells per milliliter of medium) were incubated with LH in doses of 0, .1, 1, 10, and 100 ng/ml for 7 d. Conditioned media were collected on d 1, 3, 5, and 7 of incubation. Across all days of incubation, media from small luteal cells stimulated proliferation of endothelial cells. Media from large luteal cell incubations, however, secreted an endothelial mitogen only on d 7 of culture. Mixed luteal cell cultures secreted mitogenic activity on d 3, 5, and 7 of incubation, but not on d 1. Luteinizing hormone did not influence release of mitogenic activity by any luteal cell fraction. Across all days of incubation, media from large luteal cells contained more progesterone than those from small luteal cells (528 +/- 137 vs 48 +/- 16 ng/ml with no LH). Mixed (non-elutriated) and small luteal cells increased progesterone secretion in response to LH, and this response was maintained during long-term culture. Large luteal cells did not increase progesterone secretion in response to LH. Steroidogenic activity of all cell types decreased as incubation time progressed.(ABSTRACT TRUNCATED AT 250 WORDS)

Weight of placental tissues of cows increased exponentially from Day 100 to Day 250 of gestation, but at much slower relative and absolute rates than fetal weight. In addition, growth rate of fetal placental tissues was less than that of maternal placental tissues. Concentrations of DNA, RNA and protein, however, increased in fetal placental but not in maternal placental tissues. Fetal placental tissues therefore exhibited hyperplasia, which probably contributes to increased functional capacity of the placenta during late gestation. The rate of O2 uptake in vitro was greatest for maternal placental tissues, suggesting that the maternal portion of the placenta accounts for most of the large rate of placental O2 utilization in vivo. Compared with other placental tissues, rate of secretion of macromolecules by intercaruncular endometrium was high, but decreased from Day 100 to 250, suggesting that uterine glandular secretory activity may decrease as gestation advances. Rate of secretion of macromolecules also was high for intercotyledonary tissues and increased with day of gestation, suggesting a role for secretory products of chorioallantois in gravid uterine function.