Luteal ANGPT-TIE system during selected stages of pregnancy, and normal and antigestagen-induced luteolysis in the dog.

Abstract: Rapid establishment of a vascular network is essential for normal functionality of the corpus luteum (CL). The early luteal phase is associated with increased expression of the VEGF system in canine CL. Acting in synchrony with angiopoietins (ANGPTs), VEGF system plays major roles in stabilization of blood vessels. However, the expression of the ANGPT system has not yet been investigated in the dog. Therefore, here, we investigated the luteal expression of ANGPT1, -2, and of their receptors TIE1 and -2, in pregnant dogs at selected time points during pregnancy and at normal and antigestagen-induced luteolysis. Additionally, luteal cells from early CL were incubated with PGE2 and its effects on the ANGPT system were assessed. Whereas the luteal ANGPT1 was stable until mid-gestation, TIE1 was elevated post-implantation, their expression decreased toward prepartum luteolysis. The ANGPT2- and TIE2-mRNA did not vary during pregnancy. The ANGPT2/ANGPT1 ratio was elevated during prepartum luteolysis. PGE2 increased ANGPT2, but suppressed ANGPT1 levels. None of the ANGPT-system members was affected by antigestagen treatment in mid-pregnancy. Localization of ANGPT1 was predominantly found in the tunica intima and media of vessels and ANGPT2 stained strongly in luteal cells. Both ANGPTs were localized in macrophages. TIE1 stained in the vascular tunica media, in luteal cells and macrophages, whereas TIE2 was colocalized with ANGPT1 in vascular components. In conclusion, high expression of ANGPT1 during the increased presence of VEGFA in early canine CL implies its contribution to vascular network development. The upregulation of the ANGPT2/ANGPT1 ratio during prepartum luteolysis indicates involvement of the ANGPT system in PGF2α-mediated vascular destabilization.

Angiopoietin expression in ovine corpora lutea during the luteal phase: Effects of nutrition, arginine and follicle stimulating hormone.

The aim of this study was to evaluate angiopoietin (ANGPT) 1 and 2, and tyrosine-protein kinase receptor 2 (TIE2) expression in the corpora lutea (CL) of FSH-treated, or non-treated sheep administered arginine (Arg) or vehicle (saline, Sal), and fed a control (C), excess (O) or restricted (U) diet. Ewes from each dietary group were treated with Arg or Sal (experiment 1), and with FSH (experiment 2). Luteal tissues were collected at the early-, mid- and/or late-luteal phases of the estrous cycle. Protein and mRNA expression was determined using immunohistochemistry followed by image analysis, and quantitative RT-PCR, respectively. The results demonstrated that ANGPT1 and TIE2 proteins were localized to luteal capillaries and endothelial cells of larger blood vessels, and ANGPT2 was localized to tunica media of larger blood vessels. TIE2 protein was also present in luteal cells. In experiment 1, ANGPT1 protein expression was greater in O than C during early- and mid-luteal phases, and was greatest during late-luteal phase, less at the mid- and least at the early-luteal phase; 2) TIE2 protein expression was greatest at the mid-, less at the early- and least at the late-luteal phase; 3) ANGPT1 and 2 mRNA expression was greater at the mid- and late- than the early-luteal phase, and TIE2 mRNA expression was greatest at the late-, less at the mid- and least at the early-luteal phase. The ANGPT1/2 ratio was less at the early- than mid- or late-luteal phases. In experiment 2, ANGPT1 protein expression was greater in O during the mid-luteal phase than in other groups, and was greater at the mid- than early-luteal phase. TIE2 protein expression was highest at the mid-, less at the early- and least during the late-luteal phase. ANGPT1 and 2, and TIE2 mRNA expression was higher at the mid- than the early-luteal phase. During mid-luteal phase, ANGPT1 mRNA expression was greater in C than O and U, ANGPT2 was greatest in C, less in O and least in U, and TIE2 mRNA expression was greater in C than O and U. The ANGPT1/2 ratio was higher in U than in any other group. Comparison of FSH vs. Sal treatment effects (experiment 2 vs. experiment 1) demonstrated that FSH affected ANGPT1 and/or -2, and TIE2 protein and mRNA expression depending on luteal phase and/or diet. Thus, expression of ANGPTs and TIE2 in the CL changes during the luteal lifespan, indicating their involvement in luteal vascular formation, stabilization and degradation. Moreover, this study has demonstrated that plane of nutrition and/or FSH treatment affect the ANGPT system, and may alter luteal vascularity and luteal function in sheep.

Luteal function during the estrous cycle in arginine-treated ewes fed different planes of nutrition.

Functions of corpus luteum (CL) are influenced by numerous factors including hormones, growth and angiogenic factors, nutritional plane and dietary supplements such as arginine (Arg), a semi-essential amino acid and precursor for proteins, polyamines and nitric oxide (NO). The aim of this study was to determine if Arg supplementation to ewes fed different planes of nutrition influences: (1) progesterone (P4) concentrations in serum and luteal tissue, (2) luteal vascularity, cell proliferation, endothelial NO synthase (eNOS) and receptor (R) soluble guanylate cyclase ß protein and mRNA expression and (3) luteal mRNA expression for selected angiogenic factors during the estrous cycle. Ewes (n = 111) were categorized by weight and randomly assigned to one of three nutritional planes: maintenance control (C), overfed (2× C) and underfed (0.6× C) beginning 60 days prior to onset of estrus. After estrus synchronization, ewes from each nutritional plane were assigned randomly to one of two treatments: Arg or saline. Serum and CL were collected at the early, mid and late luteal phases. The results demonstrated that: (1) nutritional plane affected ovulation rates, luteal vascularity, cell proliferation and NOS3, GUCY1B3, vascular endothelial growth factor (VEGF) and VEGFR2 mRNA expression, (2) Arg affected luteal vascularity, cell proliferation and NOS3, GUCY1B3, VEGF and VEGFR2 mRNA expression and (3) luteal vascularity, cell proliferation and the VEGF and NO systems depend on the stage of the estrous cycle. These data indicate that plane of nutrition and/or Arg supplementation can alter vascularization and expression of selected angiogenic factors in luteal tissue during the estrous cycle in sheep.

Progesterone secretion by ovine granulosa cells: effects of nitric oxide and plane of nutrition.

The aim was to evaluate the effects of nutritional plane on in vitro progesterone (P4) secretion by granulosa (G) cells cultured in the presence or absence of effectors of the nitric oxide (NO) system. Ewes were randomly assigned into three nutritional groups: control (C), overfed (O; 2 × C), or underfed (U; 0.6 × C). Follicular development was induced by FSH injections. On day 15 of the estrous cycle, G cells were isolated and cultured with or without DETA-NONOate (NO donor), L-NAME (NO synthase [S] inhibitor), Arg and (or) LH for 8 h. DETA-NONOate decreased basal and LH-stimulated P4 secretion, and L-NAME increased basal P4 secretion in all groups. In U, Arg decreased LH-stimulated P4 secretion. These data demonstrate that (i) plane of nutrition affects basal P4 secretion by G cells, (ii) the NO donor decreases, NOS inhibitor increases but Arg does not affect basal P4 secretion, and (iii) effects of Arg on LH-stimulated P4 secretion are affected by plane of nutrition in FSH-treated sheep. Thus, plane of nutrition affects G cell function, and the NO system is involved in the regulation of basal and LH-stimulated P4 secretion. The mechanism of the NO system effects on secretory activity of G cells remains to be elucidated.

Prion (PrPC) expression in ovine uteroplacental tissues increases after estrogen treatment of ovariectomized ewes and during early pregnancy.

Scrapie in sheep is spread laterally by placental transmission of an infectious misfolded form (PrPSc) of a normal prion protein (PrPC) used as a template in PrPSc formation. We hypothesized that PrPC would be expressed in uterine and placental tissues and estradiol-17ß (E2) would affect uterine PrPC expression. PrPC expression was evaluated in the uterus of long-term ovariectomized (OVX) ewes treated with an E2 implant for 2-24 h and in uteroplacental tissues from day 20 to day 30 of pregnancy. Expression of PrPC mRNA and PrPC protein increased in the uterus after E2 treatment of OVX ewes. In the maternal placenta, expression of PrPC mRNA and PrPC protein were unchanged, but in the fetal membranes (FM) PrPC mRNA and PrPC protein expression increased from day 20 to day 28. In the nonpregnant uterus, PrPC protein was immunolocalized at apical borders of the surface epithelium, in outer smooth muscle layers of large blood vessels, and in scattered stromal cells of the deep intercaruncular areas of the uterus. In the maternal placenta, PrPC protein was immunolocalized in the cytoplasm of flattened luminal epithelial cells apposed to the FM, whereas in the FM PrPC protein was in trophoblast cells and was also in several tissues of the developing embryo during early pregnancy. These data linking estrogen stimulation to increases in PrPC expression in uteroplacental tissues suggest that PrPC has a specific function during the estrous cycle and early pregnancy. Future studies should determine whether or not estrogen influences PrPC expression in other tissues, such as the nervous system and brain.

Placental development during early pregnancy in sheep: effects of embryo origin on vascularization.

Utero-placental growth and vascular development are critical for pregnancy establishment that may be altered by various factors including assisted reproductive technologies (ART), nutrition, or others, leading to compromised pregnancy. We hypothesized that placental vascularization and expression of angiogenic factors are altered early in pregnancies after transfer of embryos created using selected ART methods. Pregnancies were achieved through natural mating (NAT), or transfer of embryos from NAT (NAT-ET), or IVF or in vitro activation (IVA). Placental tissues were collected on day 22 of pregnancy. In maternal caruncles (CAR), vascular cell proliferation was less (P<0.05) for IVA than other groups. Compared with NAT, density of blood vessels was less (P<0.05) for IVF and IVA in fetal membranes (FM) and for NAT-ET, IVF, and IVA in CAR. In FM, mRNA expression was decreased (P<0.01-0.08) in NAT-ET, IVF, and IVA compared with NAT for vascular endothelial growth factor (VEGF) and its receptor FLT1, placental growth factor (PGF), neuropilin 1 (NP1) and NP2, angiopoietin 1 (ANGPT1) and ANGPT2, endothelial nitric oxide synthase 3 (NOS3), hypoxia-inducible factor 1A (HIF1A), fibroblast growth factor 2 (FGF2), and its receptor FGFR2. In CAR, mRNA expression was decreased (P<0.01-0.05) in NAT-ET, IVF, and IVA compared with NAT for VEGF, FLT1, PGF, ANGPT1, and TEK. Decreased mRNA expression for 12 of 14 angiogenic factors across FM and CAR in NAT-ET, IVF, and IVA pregnancies was associated with reduced placental vascular development, which would lead to poor placental function and compromised fetal and placental growth and development.

Placental vascular defects in compromised pregnancies: effects of assisted reproductive technologies and other maternal stressors.

Many factors negatively affect pregnancy establishment and subsequent fetal growth and development, including maternal factors such as nutritional stress, age, body mass index, and genetic background, and external factors including environmental stress, psychosocial stress, multiple fetuses, medical conditions (e.g., polycystic ovary syndrome), lifestyle choices (e.g., alcohol consumption, smoking), and assisted reproductive technologies. These same factors have similar consequences for placental growth and development, including vascular development. We and others have shown that placental vascular development begins very early in pregnancy and determines, to a large extent, placental function-that is, the magnitude of the increase in placental blood flow and thus nutrient transport to the fetus. During the peri-implantation period and also later in pregnancy, cloned (somatic cell nuclear transfer) embryos exhibit a variety of placental defects including reduced vascularization and altered expression of angiogenic factors. Although placental defects are less pronounced in pregnancies resulting from the transfer of in vitro fertilized embryos, we and others have recently demonstrated that vascularization, expression of angiogenic factors, sex steroid receptors, several epigenetic markers, and growth of utero-placental tissues all were altered during early pregnancy after transfer of embryos obtained through natural mating, in vitro fertilization, or other assisted reproductive techniques. These observations are in agreement with the recent reports that in humans even singleton pregnancies established with assisted reproductive techniques are at increased risk of preterm delivery and low birth weight, and seem especially relevant considering the rapidly expanding use of these techniques in humans and animals.

The effects of follicle stimulating hormone (FSH)-induced controlled ovarian hyperstimulation and nutrition on implantation-related gene expression in caruncular tissues of non-pregnant sheep.

Disturbances at the conceptus-maternal interface can have detrimental effects on pregnancy outcome. Additionally, changes in body condition and exogenously administered gonadotropins could affect ovarian and uterine function, including cell proliferation and ovulation rates, and alter endometrial receptivity. In ruminants, endometrial caruncles maintain placental function via interaction with fetal chorionic cotyledons. Here, the effects of feeding regimens on the expression of selected genes known to be involved in uterine receptivity were investigated in the caruncles of control and FSH-superovulated ewes. Sheep were grouped according to their diet: control fed (CF), overfed (OF) or underfed (UF), and were either superovulated with FSH (SOV) or untreated (CON, naturally cycling) (n = 3-5/group). Caruncular samples for the assessment of the transcript levels of 11 target genes were collected at either the early (day 5) or mid-luteal (day 10) phases of the luteal lifespan, resulting in 12 groups of animals. The day of the estrous cycle affected the expression of ITGAV, ITGB3, FGF10 and IGFBP3 mRNA. There was lower expression of MUC1, and higher expression of FGF10, ITGB3 and FN1, on day 10 in CF_SOV animals. Compared with CF, expression of integrins (ITGB3, ITGA5 and ITGA4) was higher in OF and UF, and higher transcript levels of HGF and IGFBP3 in UF animals on day 10. Expression of ITGA5, ITGB1, -3, -5 and MUC1 was greater in OF_SOV than CF_SOV at day 10. In conclusion, it appears that imbalanced nutrition, by altering the expression of genes responsible for intercellular communication, cell adhesion, and encoding for growth factors, could affect the uterine responsiveness to exogenously applied hormonal stimulation and, likely, uterine receptivity.

Barbed compared with standard suture: effects on cellular composition and proliferation of the healing wound in the ovine uterus.

OBJECTIVE: To compare cellular composition (fibroblasts vs. smooth muscle cells) and proliferation in uterine healing wounds after application of barbed compared with standard suture in a sheep model. DESIGN: Randomized trial (Canadian Task Force classification I) using each animal as its own control. SETTING: Certified animal research facility. Population or sample. 23 non-pregnant ewes. METHODS: A myometrial incision was created with the harmonic scalpel in each horn of the bicornuate uterus. The incisions were randomly allocated to be closed using either polyglactin 210 (Vicryl®) or barbed suture. Three months later, uterine tissues were collected, fixed and used for determination of cellular composition and proliferation using histochemistry (Masson trichrome staining) and immunohistochemistry (staining of smooth muscle cell actin and Ki67, a marker of proliferating cells) followed by image analysis. MAIN OUTCOME MEASURES: Evaluation and comparison of the cellular composition and proliferation of uterine wounds after application of barbed vs. standard suture. RESULTS: The ratio between connective tissue elements and smooth muscle cells, expression of smooth muscle cell actin and labeling index were similar in wounds after application of barbed compared with standard suture, but were different (p < 0.0001-0.05) in wounds than in non-wounded areas in uterus. CONCLUSION: Both barbed and standard sutures had similar effects on cellular composition and proliferation of uterine wounds in an animal model.

Plane of nutrition and FSH-induced superovulation affect the expression of steroid hormone receptors and growth factors in caruncular tissue of non-pregnant sheep.

Implantation is a critical step in the establishment of pregnancy and an important part of embryo-maternal contact. Uterine receptivity can be affected by changes in body condition and the maternal endocrine milieu, including those caused by the use of exogenous gonadotropins in controlled ovarian hyperstimulation to induce the development of multiple follicles. This study demonstrates the effects of FSH-mediated ovarian hyperstimulation on the caruncles of ewes under various feeding regimes. Sheep were classified into 3 categories: control fed (CF), overfed (OF), or underfed (UF). In each group, animals were superovulated with FSH or injected with a saline solution (non-treated control). Uterine caruncles were collected at the early (d 5) and mid-luteal phase (d 10) of the estrous cycle. The transcript levels of steroid hormone receptors (ESR1, ESR2, PGR) and growth factors (IGF1, IGF2, VEGFA) were investigated and their expression localized by immunohistochemical staining. As for the main findings, day of the estrous cycle affected expression of ESR1, IGF1 and IGF2, but not of ESR2, PGR and VEGFA; both feeding and superovulation had modulatory effects, with feeding (UF/OF) stimulating expression of all genes studied, and superovulation altering expression of some genes, eg IGF1, PGR and ESR1 and ESR2, in CF animals. Similarly, feeding (UF/OF) altered responsiveness to superovulation for PGR on d 5 and ESR1/ESR2 on d 5 and/or 10. Our data emphasize possible effects of dietary and/or hormonal stimuli on uterine physiology, which may affect pregnancy outcomes by disrupting uterine functionality.

Placental development during early pregnancy in sheep: cell proliferation, global methylation, and angiogenesis in the fetal placenta.

To characterize early fetal placental development, gravid uterine tissues were collected from pregnant ewes every other day from day 16 to 30 after mating. Determination of 1) cell proliferation was based on Ki67 protein immunodetection; 2) global methylation was based on 5-methyl-cytosine (5mC) expression and mRNA expression for DNA methyltransferases (DNMTs) 1, 3a, and 3b; and 3) vascular development was based on smooth muscle cell actin immunolocalization and on mRNA expression of several factors involved in the regulation of angiogenesis in fetal membranes (FMs). Throughout early pregnancy, the labeling index (proportion of proliferating cells) was very high (21%) and did not change. Expression of 5mC and mRNA for DNMT3b decreased, but mRNA for DNMT1 and 3a increased. Blood vessels were detected in FM on days 18-30 of pregnancy, and their number per tissue area did not change. The patterns of mRNA expression for placental growth factor, vascular endothelial growth factor, and their receptors FLT1 and KDR; angiopoietins 1 and 2 and their receptor TEK; endothelial nitric oxide synthase and the NO receptor GUCY13B; and hypoxia inducing factor 1 α changed in FM during early pregnancy. These data demonstrate high cellular proliferation rates, and changes in global methylation and mRNA expression of factors involved in the regulation of DNA methylation and angiogenesis in FM during early pregnancy. This description of cellular and molecular changes in FM during early pregnancy will provide the foundation for determining the basis of altered placental development in pregnancies compromised by environmental, genetic, or other factors.

Barbed vs standard suture: randomized single-blinded comparison of adhesion formation and ease of use in an animal model.

STUDY OBJECTIVE: To compare adhesion formation and ease of use of barbed vs traditional suture during myometrial closure in a sheep model. DESIGN: Randomized trial using each subject as its own control (Canadian Task Force classification I). SETTING: Certified animal research facility. SUBJECTS: Twenty-three nonpregnant ewes. INTERVENTIONS: The Harmonic scalpel was used to create a myometrial incision in each uterine horn, and the incisions were randomly allocated to be closed using either polyglactin 210 (Vicryl) or barbed suture. Each animal served as its own control, with 1 horn sutured using barbed suture and the other horn sutured using Vicryl suture. Ease of use was evaluated by comparing closure times. Adhesion formation was compared at necropsy 3 months later using a standardized adhesion-formation scoring system. The evaluator of the adhesion score was blinded to the exposure at surgery. MEASUREMENTS AND MAIN RESULTS: Mean total procedure time was 13.3 minutes. Myometrial closure was significantly faster using barbed vs traditional suture (126.5 seconds vs 272.6 seconds; p <.001). At necropsy 3 months later, adhesions were observed in 12 uterine horns (52.2%) in the barbed suture group vs 10 uterine horns (43.5%) in the Vicryl group (p = .62). The mean (SD) adhesion score was not significantly different between the barbed suture group (3.78 [3.92]) vs the Vicryl group (3.04 [3.75]) ( p = .16). CONCLUSION: Barbed suture significantly facilitates myometrial closure and is associated with adhesion formation and adhesion severity that is not different from that using Vicryl in an animal model.

Mid- to late-gestational maternal nutrient restriction followed by realimentation alters development and lipid composition of liver and skeletal muscles in ovine fetuses.

Maternal nutrient restriction during gestation adversely affects offspring growth and development of liver and skeletal muscle tissues. Realimentation following nutrient restriction may alleviate these negative impacts on development but may alter metabolism and tissue composition. Forty-eight ewes, pregnant with singletons, were fed to meet 100% National Research Council (NRC) recommendations starting at the beginning of gestation. On day 50 of gestation, seven ewes were euthanized (BASE), and fetal liver, skeletal muscles, and blood samples were collected. The remaining animals were fed either 100% of NRC recommendations (CON) or 60% NRC recommendations (RES), a subset were euthanized at day 90 of gestation (n = 7/treatment), and fetal samples were collected. Remaining ewes were maintained on the current diet (CON-CON, n = 6; RES-RES, n = 7) or switched to the alternate diet (CON-RES, RES-CON; n = 7/treatment). On day 130 of gestation, the remaining ewes were euthanized, and fetal samples were collected. At day 130 of gestation, maternal nutrient restriction during late-gestation (RES-RES and CON-RES) decreased fetal liver weight (P < 0.01) and cross-sectional area in triceps brachii (P = 0.01; TB), longissimus dorsi (P = 0.02; LM), and semitendinosus (P = 0.05; STN) muscles. Maternal nutrient restriction during mid-gestation increased hepatocyte vacuole size at day 130 of gestation. Late-gestational maternal nutrient restriction increased mRNA expression of insulin-like growth factor (IGF) binding protein-1 (P < 0.01), glycogen synthase 2 (P = 0.01; GYS2), and pyruvate dehydrogenase kinase 1 (P < 0.01; PDHK1) in the liver and IGF receptor 1 (P = 0.05) in the LM. Lipid concentration in the LM was decreased by late-gestational nutrient restriction (P = 0.01) and increased by mid-gestational nutrient restriction in STN (P = 0.03) and TB (P < 0.01). Principal component analysis of lipidomics data demonstrated clustering of principal components by day of gestation and elastic net regression identified 50, 44, and 29 lipids that classified the treatments in the fetal liver, LM, and blood, respectively. In conclusion, restricting maternal nutrition impacts fetal liver and muscle morphology, gene expression, and lipid metabolism, whereas realimentation attenuated some of these effects. Therefore, realimentation may be a viable strategy to reduce the impacts of nutrient restriction, but can lead to alterations in lipid metabolism in sheep.

Markers of ovarian antral follicular development in sheep: comparison of follicles destined to ovulate from the final or penultimate follicular wave of the estrous cycle.

Treatment of non-prolific western white-faced ewes with prostaglandin F(2α) (PGF(2α)) and medroxyprogesterone acetate (MAP) increases the ovulation rate as a result of ovulations from the penultimate wave in addition to the final wave of the cycle. The objective of the current study was to evaluate the expression of markers of vascularization/angiogenesis, a marker of intercellular communication, and cellular proliferation and apoptosis in follicles from the penultimate and final waves. On day 8 of the estrous cycle, 15 ewes were administered a single injection of PGF(2α) and an intravaginal MAP sponge, which remained in place for 6 days. Two days after sponge removal, ovaries which contained follicles from the penultimate and final waves were collected and processed for immunohistochemistry followed by image analysis, and for quantitative real-time RT-PCR. Expression of factor VIII (marker of vascularization), proliferating cell nuclear antigen, and GJA1 (Cx43; marker of gap junctional communication) was greater (P<0.05) in follicles from the final wave compared with follicles from the penultimate wave. For theca cells, mRNA expression for vascular endothelial growth factor (VEGF) was greater (P<0.05) and tended to be greater (P≤0.1 and ≥0.05) for GJA1 and endothelial nitric oxide synthase in follicles from the final wave compared with follicles from the penultimate wave. For granulosa cells, the mRNA expression for GJA1 was greater (P<0.05) and tended to be greater (P≤0.1 and ≥0.05) for VEGF in follicles from the final wave compared with follicles from the penultimate wave. In conclusion, extension of the lifespan of follicles in the penultimate wave reduces follicular viability in the ewe.

Placental development during early pregnancy in sheep: vascular growth and expression of angiogenic factors in maternal placenta.

Placental vascular development (angiogenesis) is critical for placental function and thus for normal embryonic/fetal growth and development. Specific environmental factors or use of assisted reproductive techniques may result in poor placental angiogenesis, which may contribute to embryonic losses and/or fetal growth retardation. Uterine tissues were collected on days 14, 16, 18, 20, 22, 24, 26, 28, and 30 after mating and on day 10 after estrus (nonpregnant controls) to determine vascular development and expression of several factors involved in the regulation of angiogenesis in the endometrium. Compared with controls, several measurements of endometrial vascularity increased (P<0.001) including vascular labeling index (LI; proportion of proliferating cells), the tissue area occupied by capillaries, area per capillary (capillary size), total capillary circumference per unit of tissue area, and expression of factor VIII (marker of endothelial cells), but capillary number decreased (P<0.001). Compared with controls, mRNA for placental growth factor, vascular endothelial growth factor receptors, angiopoietins (ANGPT) 1 and 2, ANGPT receptor TEK, endothelial nitric oxide synthase, and hypoxia-inducible factor 1alpha increased (P<0.05) during early pregnancy. Vascular LI was positively correlated (P<0.05) with several measurements of vascularity and with mRNA expression of angiogenic factors. These data indicate that endometrial angiogenesis, manifested by increased vascularity and increased expression of several factors involved in the regulation of angiogenesis, is initiated very early in pregnancy. This more complete description of early placental angiogenesis may provide the foundation for determining whether placental vascular development is altered in compromised pregnancies.

Changes in cell proliferation, but not in vascularisation are characteristic for human endometrium in different reproductive failures--a pilot study.

BACKGROUND: Reproductive failure, determined as recurrent spontaneous abortions (RSA) or recurrent implantation failure (RIF) in women is not well understood. Several factors, including embryo quality, and cellular and molecular changes in endometrium may contribute to the insufficient feto-maternal interaction resulting in reproductive failure. Prior clinical studies suggest an inadequate endometrial growth and development of the endometrium, leading to a lesser endometrial thickness. METHODS: We therefore aimed to determine the cellular proliferation using Ki67, and the expression of markers of vascularisation, such as factor VIII (a marker of endothelial cells) and smooth muscle cell actin (SMCA; a marker of pericytes and smooth muscle cells) in endometrium of healthy women and women with RSA or RIF. LH-dated mid-secretory endometrial biopsies of seven healthy women and twenty women with reproductive failure were examined via immunohistochemistry followed by image analysis. RESULTS: Cellular proliferation but not expression of factor VIII or SMCA was decreased (P < 0.0004) in endometrium of women with RSA and RIF compared to healthy controls. CONCLUSION: Our data indicate that reproductive failure is due to insufficient cell proliferation/tissue growth rather than inadequate vascularisation in the endometrium.

Nuclear and membrane progesterone receptors expression in placenta from early to late pregnancy in sheep: Effects of restricted nutrition and realimentation.

Nutrient restriction and/or realimentation may affect several placental functions, such as expression of selected regulatory factors, blood flow and other processes in sheep and other species. To determine the effects of the plane of nutrition, nulliparous white face ewes (6-8 months) carrying singletons on day 50 of gestation were randomly assigned to two dietary treatments receiving 100% of National Research Council recommendations (control; C) or 60% of C (restricted; R). Two groups remained on C or R diets from day 50 until day 130. From day 90-130 another group of C fed ewes was switched to the R diet, and another group of R fed ewes was switched to the C diet. This resulted in 7 groups (n = 5-6 ewes/group): C (day 50, 90 and 130), R (day 90 and 130), CR (day 130) and RC (day 130). At these time points, placental tissues were collected for the evaluation of progesterone receptor (PGR) protein expression (whole tissue), and mRNA expression in maternal (caruncular, CAR) and fetal (cotyledon, COT) (separated tissues). Data were statistically analyzed using analysis of variance (SAS 9.4). Protein for PGRAB and PGRB isoforms was detected using immunohistochemistry in all placental tissues, but the pattern of expression differed depending on pregnancy stage and placental compartment (e.g., CAR vs COT). PGRAB protein expression, quantified using image analysis, was greater (P < 0.04) on day 50 than 90 or 130, and was not affected by plane of nutrition. In CAR and COT, PGRAB mRNA expression was greater (P < 0.05) on day 50 than 90 or 130. PGRB mRNA expression was greater (P < 0.03) in CAR on day 50 than 90 and 130, and was greatest (P < 0.02) in COT on day 50, less on day 130, and least on day 90. For the membrane progesterone receptors, PAQR7 (membrane PGR alpha) mRNA expression was greater (P < 0.05) on days 50 and 90 than 130 in CAR, and greater (P < 0.01) on days 50 than 90 and 130 in COT; PAQR8 (membrane PGR beta) was similar throughout pregnancy in CAR and COT, and PAQR5 (membrane PGR gamma) was greatest (P < 0.0001) on day 130 in COT, but similar throughout pregnancy in CAR. Plane of nutrition affected (P < 0.05) mRNA expression for all genes in CAR and COT throughout pregnancy. These data indicate that expression of PGR in ovine placenta is dependent on stage of pregnancy and plane of nutrition in sheep. The mechanisms of how diet and stage of pregnancy influences placental PGR expression and function remains to be elucidated.

Maternal periconceptual nutrition, early pregnancy, and developmental outcomes in beef cattle.

The focus of this review is maternal nutrition during the periconceptual period and offspring developmental outcomes in beef cattle, with an emphasis on the first 50 d of gestation, which represents the embryonic period. Animal agriculture in general, and specifically the beef cattle industry, currently faces immense challenges. The world needs to significantly increase its output of animal food products by 2050 and beyond to meet the food security and agricultural sustainability needs of the rapidly growing human population. Consequently, efficient and sustainable approaches to livestock production are essential. Maternal nutritional status is a major factor that leads to developmental programming of offspring outcomes. Developmental programming refers to the influence of pre-and postnatal factors, such as inappropriate maternal nutrition, that affect growth and development and result in long-term consequences for health and productivity of the offspring. In this review, we discuss recent studies in which we and others have addressed the questions, "Is development programmed periconceptually?" and, if so, "Does it matter practically to the offspring in production settings?" The reviewed studies have demonstrated that the periconceptual period is important not only for pregnancy establishment but also may be a critical period during which fetal, placental, and potentially postnatal development and function are programmed. The evidence for fetal and placental programming during the periconceptual period is strong and implies that research efforts to mitigate the negative and foster the positive benefits of developmental programming need to include robust investigative efforts during the periconceptual period to better understand the implications for life-long health and productivity.

Cellular proliferation and vascularization in ovine fetal ovaries: effects of undernutrition and selenium in maternal diet.

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

Lipopolysaccharide disrupts gap junctional intercellular communication in an immortalized ovine luteal endothelial cell line.

Gram-negative bacteria, in particular Escherichia coli with its cell wall lipopolysaccharide (LPS), often cause metritis and mastitis in domestic animals. Ovarian LPS accumulation may initiate local inflammatory reactions mediated through cell surface Toll-like receptors (TLRs). This may disrupt ovarian functionality leading to infertility. Possible adverse effects of LPS on luteal activity are not yet well explored. We hypothesized that LPS could lead to alterations in luteal vascular functionality. Therefore, we established an in vitro cell line model (OLENDO) by immortalizing microvascular endothelial cells isolated from ovine corpus luteum (CL) with a potent Simian Virus 40 T-antigen (SV40-Tag). OLENDO exhibit endothelial cell characteristics, like low-density lipoprotein (LDL) uptake, express BSL-I, and VEGFR2, as well as TLR2 and TLR4 receptors. LPS-treatment of OLENDO altered in vitro tube formation, had no effects on cell viability and decreased gap junctional intercellular communication (GJIC). LPS did not impair GJA1/Cx43 protein expression, but altered its cellular localization showing signs of internalization. Taken together, we demonstrated the mechanisms underlying LPS induced impairment of luteal GJIC and immune processes in a novel and well-characterized OLENDO cell line.