Timing and duration of nutrient restriction and its impacts on placental development and umbilical blood flow in adolescent sheep.

We hypothesized that nutrient restriction from day 50-90 of gestation decreases umbilical blood flow and that umbilical blood flow would recover to control values upon realimentation during late gestation (d 90 to 130) or remain reduced in ewes that continued to be nutrient restricted. On d 50 of gestation, young nulliparous whiteface ewes (6-8 mo; n = 41) carrying singletons were randomly assigned to two dietary treatments: 100% of NRC recommendations (CON) or 60% of CON (RES). On d 90 of gestation, ewes either remained on CON or RES until d 130, or CON ewes were RES from d 90 to 130, or RES ewes were realimented to CON from d 90 to 130. This resulted in 4 treatment groups on day 130: CON-CON, CON-RES, RES-RES, RES-CON. Umbilical blood flow and fetal and placental measurements were obtained via ultrasonography every 10 days from day 50-110. Non-survival surgeries were performed on days 50, 90, and 130 (n = 6-7 ewes/group) where uterine artery and umbilical blood flows were measured during surgery via ultrasonography. Conceptus weights were recorded and placentomes collected to determine binucleate cell numbers. The study was conducted as a completely randomized design arrangement with repeated measures. Data were analyzed using the MIXED procedure of SAS. There was a nutritional treatment by day interaction (P < 0.01) with CON ewes having greater umbilical blood flow compared with RES by d 90. Fetal biparietal distance, abdominal width, and kidney area increased (P < 0.05) in CON-RES with all these measurements increasing during late gestation. We partially accept our hypothesis as nutrient restriction during mid gestation decreased umbilical blood flow. However, blood flow did not return to control levels upon realimentation. By d 130, fetal and placental weights were similar between RES-RES and CON-CON. Binucleate cell numbers in the fetal trophoblast were not influenced by nutritional treatments. Our findings suggest that refeeding previously nutrient restricted pregnant adolescent ewes to control levels does not reestablish umbilical blood flow. Adequate placental development during mid gestation could protect the fetus from a decreased umbilical blood flow later in gestation when nutrients were limited by 40%.

Uterine kallikrein and arterial bradykinin activities and uterine arterial proliferation in response to acute estradiol-17ß exposure in ovariectomized ewes.

Estradiol-17ß (E2) increases kallikrein in rodent and human reproductive tissues. Kallikrein specific activity is increased in the porcine uterus when conceptus E2 is secreted at maternal recognition of pregnancy. When kallikrein acts on kininogen to liberate bradykinin, angiogenic and vasoactive factors are released. The uterus of ovariectomized ewes administered E2 undergoes rapid vascular changes via different patterns of angiogenic and vasoactive factors. Our hypothesis was that E2 would increase the specific activity and protein secretion of tissue kallikrein in endometrial explants culture media (ECM) and ewes exposed to E2 would have uterine arteries that would be more sensitive to the vasodilatory effects of bradykinin. Ovariectomized ewes received 100 mg of E2 implants for 0, 12, 24, or 48 h. After treatment, uterine weights were determined, and caruncles were processed for ECM. Uterine weights and uterine weight per ewe body weight were significantly greater in the 12 and 24 h ewes compared with the 0 h ewes, with the 48 h ewes being similar to the 24 h ewes. There were no statistically significant differences in caruncular tissue kallikrein protein secretion among the treatment groups. There was a tendency (P = 0.09) for duration of E2 exposure to influence tissue kallikrein specific activity where kallikrein activity was greater (P ≤ 0.05) in the 12 and 48 h ewes compared with the 0 h ewes, with 24 h ewes being intermediate (unprotected F test). Uterine arteries from ewes with E2 for 24 and 48 h had more sensitivity to bradykinin, via the bradykinin receptor 2, than uterine arteries from ewes with 0 or 12 h E2 exposure. We fail to reject our hypothesis as E2 did elicit a positive response in tissue kallikrein specific activity and bradykinin response. Further investigations are needed to determine how kallikrein and bradykinin may be involved in vascular remodeling of the ovine uterus.

Acute effects of estradiol-17ß on plasma volume and uterine cell proliferation in sheep.

Ovarian steroids play an important role in increasing plasma volume in pregnant females and preparing the uterus for implantation. We hypothesized that a short duration of increased estradiol-17ß (E2) would increase plasma volume and uterine cell proliferation in ovariectomized ewes. Adult non-pregnant Romanov ewes (n = 15) were ovariectomized. After ovariectomy, ewes were individually housed and were offered water at ad libitum intake and were fed a pelleted diet at maintenance once daily according to body weight. After at least 30 days post-ovariectomy ewes were fasted and received an implant placed in the axillary region that contained 100 mg of E2 (E2; n = 8) or a sham implant with no E2 (CON, n = 7). After 24 h, ewes were weighed prior to plasma volume measurement procedures. Plasma volume was determined using the Evans blue dye method. Blood samples were taken at 0 (pre dye injection), 5, 10, 15, 20, 25, 30, 40, 50 and 60 min after dye injection. After the final blood collection, ewes were euthanized with an overdose of sodium pentabarbital and uterine weights were recorded. Uterine cross-sections were fixed in formalin for immunohistochemical localization of Ki67 (a marker of proliferating cells) followed by image generation of luminal epithelium and endometrial stroma (5 areas each/tissue section) and analysis to determine the proportion of proliferating cells. Plasma volume tended to be greater in E2 vs CON (2.75 ± 0.11 vs. 2.54 ± 0.12 L, P = 0.07) and uterine weights were greater in E2 vs CON (27.25 ± 2.35 vs. 17.35 ± 2.51 g, P < 0.01). Water intake after implant placement was similar in E2 and CON (3.85 vs. 4.87 ± 0.67 L; P = 0.28). Cell proliferation in the luminal epithelium was greater in E2 vs CON (6.55 vs. 1.2 ± 1.75%, P = 0.02) and stromal cells tended to be greater in E2 vs CON (0.59 vs 0.37 ± 0.06%, P = 0.07). Our results demonstrate that E2-treatment tends to increase plasma volume acutely and increases uterine cell proliferation in ewes.