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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.

Both maternal and fetal genotypes contribute to conceptus development. The objective was to determine how placentome number, size, and type and fetal weight was influenced after reciprocal embryo transfer in Columbia and Romanov sheep. Reciprocal embryo transfer was conducted between Columbia and Romanov ewes where a single embryo was transferred into Romanov and Columbia recipients [Romanov embryo in a Romanov uterus (RinR, n = 9); Romanov embryo in a Columbia uterus (RinC, n = 7); Columbia embryo in a Columbia uterus (CinC, n = 8); Columbia embryo in a Romanov uterus (CinR, n = 4)]. On day 130 of gestation, fetuses were weighed and placentomes were morphologically typed, weighed, and measured. Regardless of maternal genotype, Romanov fetuses were smaller (P

To determine the effects of maternal nutrient restriction and re-alimentation on fetal liver and muscle development, 48 pregnant ewes with singletons, were fed a control diet [100% National Research Council (NRC) requirements (CON)] starting at the beginning of gestation. On day 50 of gestation, ewes (n = 7) were euthanized and fetal liver and skeletal muscle samples were collected. The remaining animals were fed either CON or 60% NRC requirements (RES), a subset were euthanized at day 90 of gestation (n = 7/treatment), and fetal samples obtained. Remaining ewes were maintained on the current diet (CON-CON, n = 6; RES-RES, n = 7) or switched to alternative diet (CON-RES, RES-CON; n = 7/treatment). On day 130 of gestation, remaining ewes were euthanized, and fetal samples collected. Fetal liver was analyzed for general tissue morphology, and fetal skeletal muscles were analyzed for lipid accumulation. mRNA expression of growth and metabolic factors were quantified in liver and muscle tissues. Hepatocellular vacuolation was increased in RES-CON and RES-RES compared with CON-CON and CON-RES (P < 0.01). In semitendinosus and triceps brachii, intramyocellular lipid content increased 19% and 15%, respectively, in RES-CON and RES-RES compared with CON-CON and CON-RES (P£0.02) and in longissimus dorsi, lipid content was decreased 7% in CON-RES and RES-RES compared with CON-CON and RES-CON (P=0.01). In liver, insulin-like growth factor binding protein-1, glycogen synthase 2, and pyruvate dehydrogenase kinase 1 expression increased 1.92-fold, 1.45-fold, and 1.47-fold, respectively (P£0.03) in CON-RES and RES-RES compared with RES-CON and CON-CON. In LD, IGF1-R expression increased 3.19-fold in CON-RES and RES-RES compared with RES-CON and CON-CON (P = 0.05). These results demonstrate that maternal nutrient restriction followed by re-alimentation restores liver and muscle gene expression of growth and metabolic factors while negatively impacting liver composition and muscle lipid content potentially leading to altered tissue function and metabolism later in life. Supported by USDA-AFRI grants 2016-67016-24884 and 2017-67016-26568.

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.

Estradiol-17β (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.

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.

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-17β (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.

To determine the effects of maternal nutrient restriction and re-alimentation on the fetal liver proteome, 48 pregnant ewes with singletons were fed a control diet [100% National Research Council (NRC) requirements (CON)] starting at the beginning of gestation. On day 50 of gestation, a subset of ewes were fed either CON or 60% NRC requirements (RES). A subset of ewes were euthanized at day 90 of gestation (n = 7/treatment), and fetal liver samples collected. Remaining ewes were maintained on the current diet (CON-CON, n=6; RES-RES, n = 7) or switched to alternative diet (CON-RES, RES-CON; n=7/treatment). On day 130 of gestation, remaining ewes were euthanized, and fetal samples collected. Fetal liver proteins were extracted, digested by trypsin and subjected to multiplexed, label-based quantitative mass spectrometry analysis integrating Tandem Mass Tags. Proteins were identified and quantified using Proteome Discoverer (v2.5, Thermo Scientific) and differential abundance analysis was performed using ANOVA and post hoc Tukey’s HSD test. Hierarchal clustering analysis showed clustering of treatments by day of gestation. However, differences were also observed between treatments. At day 90 of gestation, 23 proteins were differentially expressed in RES compared with CON among which glycyl tRNA synthetase and pyruvate carboxylase were increased 12% and 10%, respectively (P < 0.03). At day 130 of gestation, 24, 5, and 71 proteins were differentially expressed in CON-RES, RES-CON, and RES-RES, respectively, compared with CON-CON. Carnosine dipeptidase 2 was decreased 7% in CON-RES and rho associated protein kinase and glycogen synthase 2 were increased 20% and 26% in RES-CON and RES-RES, respectively, compared with CON-CON (P < 0.04). These results indicate that nutrient restriction during mid- and late-gestation impacts expression of proteins involved in gluconeogenesis, glycogenesis, and the formation of carnosine, an integral molecule in beta-oxidation, and that re-alimentation alters proteins involved in cell migration pathways. Supported by USDA-AFRI grants 2016-67016-24884 and 2017-67016-26568.

To determine the effects of maternal nutrient restriction and re-alimentation on offspring metabolism, 48 pregnant ewes with singletons, were fed a control diet [100% National Research Council (NRC) requirements (CON)] starting at the beginning of gestation. On day 50 of gestation, ewes (n = 7) were euthanized and fetal liver, muscle, and blood samples were collected. The remaining animals were fed either CON or 60% NRC requirements (RES), a subset were euthanized at day 90 of gestation (n = 7/treatment), and fetal samples obtained. Remaining ewes were maintained on the current diet (CON-CON, n = 6; RES-RES, n = 7) or switched to alternative diet (CON-RES, RES-CON; n = 7/treatment). On day 130 of gestation, remaining ewes were euthanized, and fetal samples collected. Fetal liver, longissimus dorsi, and blood metabolites were analyzed using LC-MS/MS at Metabolon Inc. Pathway enrichment analysis was conducted using MetaboAnalyst 4.0. In liver, muscle, and blood, 64, 44, and 34 pathways were enriched between treatments at day 130 gestation and 10, 6, and 11 pathways were enriched at day 90 gestation, respectively. Arginine and proline metabolism; primary bile acid biosynthesis; and valine, leucine, and isoleucine biosynthesis were the most highly enriched pathways in RES compared with CON in liver, muscle, and blood, respectively. Additionally, the pentose phosphate pathway; valine, leucine, and isoleucine metabolism; and phenylalanine metabolism were the most highly enriched pathways in RES-CON compared with CON-CON in liver, muscle, and blood, respectively. In liver, ribulose 5-phosphate, xylulose 5-phosphate, and ribose 5-phosphate were decreased 1.82-, 1.54-, and 2.38-fold, respectively in RES-CON compared with CON-CON (P ≤ 0.05). Total triacylglycerols were increased 3.04-fold in muscle and decreased 1.57-fold in blood in RES-CON and RES-RES compared with CON-CON and CON-RES (P ≤ 0.05). Mid-gestational nutrient restriction and subsequent re-alimentation altered distinct metabolic amino acid, carbohydrate, and lipid pathways, potentially altering postnatal growth. Supported by USDA-AFRI grants 2016-67016-24884 and 2017-67016-26568.

During pregnancy, the placenta plays a pivotal role in fetal-maternal communication. Despite the known involvement of trophoblast mononuclear (MNC) and binuclear (BNC) cells in placentation and fetal development, the regulatory mechanisms underlying the gene expression in these cells and their role in pregnancy have not been fully elucidated. Thus, a genome-wide expression profile analysis of MNC and BNC from ovine placentomes on day 90 and 130 of pregnancy was performed to identify differentially expressed genes (DEG) and regulatory pathways. After tissue separation, enzymatic digestion, and elutriation, MNC and BNC were obtained, and the total RNA was isolated and sequenced (n = 2 ewes/day). Differential expression analysis was carried out with DESeq2 after library quality control and read mapping using FastQC and STAR, respectively. Among six pairwise contrasts possible, herein, we focused on the one between MNC and BNC on day 130. Accordingly, we identified 514 genes upregulated and 161 downregulated in BNC (adj.Pval < 0.05). Interestingly, we found nine DE long intergenic noncoding RNAs (lincRNA). Although the function of lincRNAs remains under investigation, its transcription has been associated with gene expression regulation in utero. The DEGs functional over-representation analysis included Rap1 and PI3k-Akt signaling pathways as up-regulated in BNC (adj.Pval. < 0.05). These pathways are activated by extracellular signals and are involved in cell proliferation and differentiation, morphogenesis, and energy metabolism. Furthermore, we identified PLA2G10 gene that codes a phospholipase protein involved in the production of prostaglandins, which have been associated with placental blood flow. These findings suggest an intricate and complex network underlying gene expression between cells and time points that are essential for the fetal-maternal placenta relationship. This project was supported by Agriculture and Food Research Initiative Competitive Grant n. 2016-67016-24884 from the USDA National Institute of Food and Agriculture.

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.

Kisspeptin, neurokinin B, and dynorphin are imperative for GnRH/LH pulsatility and reproductive cyclicity. Neurons co-expressing these neuropeptides, KNDy neurons, within the arcuate nucleus of the hypothalamus (ARC) are positioned to integrate energy balance signals from neuronal and glial cells. Energy balance mediates neurokinin B expression in the ARC and LH pulse amplitude. Dynorphin mediates progesterone negative feedback on GnRH neurons. The hypothesis that the number of KNDy-expressing neurons in the ARC of ewes during the luteal phase of the estrous cycle is influenced by energy balance was tested using ovary-intact, mature ewes fed to lose, maintain, or gain body weight. Fluorescent multiplex immunohistochemistry was employed to identify and quantify neurons expressing a single neuropeptide and co-expressing kisspeptin, neurokinin B, and dynorphin in the ARC. Kaminski et al. (1) reported previously that concentrations of insulin and leptin differed between ewes fed to achieve different body weights and that ewes fed to gain body weight had increased concentrations of progesterone in the luteal phase of the estrous cycle. Moreover, tanycyte density and cellular penetration into the ARC are increased in ewes fed to gain body weight (2). Number of neurons in the ARC expressing kisspeptin (14.9 ± 2.7 neurons, 20.9 ± 3.6 neurons, and 51.5 ± 3.3 neurons in ewes fed to lose, maintain, and gain body weight, respectively), neurokinin B (21.5 ± 3.2 neurons, 31.3 ± 4.3 neurons, and 56.0 ± 3.9 neurons in ewes fed to lose, maintain, and gain body weight, respectively), and dynorphin (10.1 ± 2.4 neurons, 14.9 ± 3.2 neurons, and 33.1 ± 2.9 neurons in ewes fed to lose, maintain, and gain body weight, respectively) protein was increased (P < 0.0001) in ewes fed to gain body weight. Number of KNDy neurons in the ARC expressing kisspeptin, neurokinin B, and dynorphin protein was decreased in ewes fed to lose body weight (1.0 ± 0.5 neurons; P = 0.01) and increased in ewes fed to gain body weight (6.7 ± 0.6 neurons; P = 0.0005) when compared to ewes fed to maintain body weight (3.3 ± 0.7 neurons). These findings suggest that expression of kisspeptin, neurokinin B, and dynorphin protein in the ARC during the luteal phase of the estrous cycle may be influenced by nutritionally-induced alterations in circulating concentrations of progesterone that drive changes in morphology and density of tanycytes. Moreover, these results demonstrate that changes in KNDy neurons within the ARC occur as an adaptation to energy balance, potentially regulated divergently by metabolic milieu. References: (1) Kaminski et al., Theriogenology. 2015 83:808-16. (2) Prezotto et al., Domestic Animal Endocrinology. 2020 Accepted.

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.

The aim of this study was to evaluate the pattern of protein expression of the steroid receptor isoforms of nuclear progesterone receptors (PGR) A and B, and estrogen receptors (ESR1 and 2) in utero-placental compartments during early pregnancy. Utero-placental tissues were collected from days 14–30 (n = 4 ewes/day), and uterine tissues were collected from non-pregnant ewes on day 10 after estrus (n = 4). Cross sections of formalin-fixed and paraffin embedded tissues were immunofluorescently stained to detect PGRAB, PGRB, ESR1 and ESR2, followed by image generation of entire cross-sections of uterine and utero-placental tissues, confocal imaging of individual uterine and utero-placental compartments, and image and statistical analyses. PGRAB, PGRB, ESR1 and ESR2 were detected in several compartments of uterine and utero-placental tissues. Quantitative image analysis of staining intensity demonstrated that compared to non-pregnant controls 1) expression of PGRAB and PGRB was less in luminal epithelium and endometrial glands from day 14–16 till 30; 2) PGRAB expression tended to be greater in endometrial and myometrial blood vessels on days 28 and/or 30; 3) PGRB expression in myometrum was lower on days 16 and 28; 4) ESR1 in endometrial stroma was lower in all days of pregnancy; 5) ESR2 expression was similar in all compartments and not affected by pregnancy stage; and 6) in FM, expression of steroid receptors was similar. Thus, we have demonstrated spatial and temporal expression of nuclear PGR and ESR isoforms in utero-placental compartments during early pregnancy.

To evaluate expression of progesterone receptor (PGR) AB in follicle stimulating hormone (FSH)-treated or non-treated sheep administered with arginine (Arg) or saline (Sal) fed a control (C), excess (O) or restricted (U) diet, uterine tissues were collected at the early, mid and/or late luteal phases. In exp. 1, ewes from each diet were randomly assigned to one of two treatments, Arg or Sal administration three times daily from day 0 of the first estrous cycle until uterine tissue collection. In exp. 2, ewes were injected twice daily with FSH on days 13-15 of the first estrous cycle. Uterine tissues were immunostained to detect PGR followed by image analysis. PGR were detected in luminal epithelium (LE), endometrial glands (EG), endometrial stroma (ES), myometrium (Myo), and endometrial and myometrial blood vessels. The percentage of PR-positive cells and/or intensity of staining were affected by phase of the estrous cycle, plane of nutrition, and/or FSH but not by Arg. In exp. 1, percentage of PGR-positive cells in LE and EG but not in ES and Myo was greater at the early and mid than late luteal phase, was not affected by plane of nutrition, and was similar in LE and EG. Intensity of staining was affected by phase of the estrous cycle and plane of nutrition in LE, EG and Myo, and was the greatest in LE, less in EG, and least in ES and Myo. In exp. 2, percentage of PGR-positive cells in LE, EG, ES and Myo was affected by phase of the estrous cycle, but not by plane of nutrition; was greater at the early than mid luteal phase; and was greatest in LE and EG, less in luminal (superficial) ES and Myo and least in deep ES. Intensity of staining was affected by phase of the estrous cycle and plane of nutrition in all compartments but ES, and was the greatest in LE and luminal EG, less in deep EG, and least in ES and Myo. Comparison of data for FSH (superovulated) and Sal-treated (non-superovulated) ewes demonstrated that FSH affected PR expression in all evaluated uterine compartments depending on plane of nutrition and phase of the estrous cycle. Thus, PGR are differentially distributed in uterine compartments, and PGR expression is affected by nutritional plane and FSH, but not Arg depending on phase of the estrous cycle. Such changes in dynamics of PGR expression indicate that diet plays a regulatory role and that FSH-treatment may alter uterine functions.

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. (c) 2021 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ )

The corpus luteum (CL) is an ovarian structure which is critical for the maintenance of reproductive cyclicity and pregnancy support. Diet and/or diet components may affect some luteal functions. FSH is widely used to induce multiple follicle development and superovulation. We hypothesized that FSH would affect luteal function in ewes fed different nutritional planes. Therefore, the aim of this study was to determine if FSH-treatment affects (1) ovulation rate; (2) CL weight; (3) cell proliferation; (4) vascularity; (5) expression of endothelial nitric oxide (eNOS) and soluble guanylate cyclase (sGC) proteins; and (6) luteal and serum progesterone (P4) concentration in control (C), overfed (O), and underfed (U) ewes at the early- and mid-luteal phases. In addition, data generated from this study were compared to data obtained from nonsuperovulated sheep and described by Bass et al. Ewes were categorized by weight and randomly assigned into nutrition groups: C (2.14 Mcal/kg; n = 11), O (2xC; n = 12), and U (0.6xC; n = 11). Nutritional treatment was initiated 60 d prior to day 0 of the estrous cycle. Ewes were injected with FSH on day 13-15 of the first estrous cycle, and blood samples and ovaries were collected at early- and mid-luteal phases of the second estrous cycle. The number of CL/ewe was determined, and CL was dissected and weighed. CL was fixed for evaluation of expression of Ki67 (a proliferating cell marker), CD31 (an endothelial cell marker), and eNOS and sGC proteins using immunohistochemistry and image analysis. From day 0 until tissue collection, C maintained, O gained, and U lost body weight. The CL number was greater (P 0.03) in C and O than U. Weights of CL, cell proliferation, vascularity, and eNOS but not sGC expression were greater (P0.001), and serum, but not luteal tissue, P4 concentrations tended to be greater (P = 0.09) at the early- than mid-luteal phase. Comparisons of CL measurements demonstrated greater (P0.01) cell proliferation and serum P4 concentration, but less vascularity at the early and mid-luteal phases, and less CL weight at the mid-luteal phase in superovulated than nonsuperovulated ewes; however, concentration of P4 in luteal tissues was similar in both groups. Thus, in superovulated ewes, luteal cell proliferation and vascularity, expression of eNOS, and serum P4 concentration depend on the stage of luteal development, but not diet. Comparison to control ewes demonstrated several differences and some similarities in luteal functions after FSH-induced superovulation.

Satellite cells are muscle stem cells that contribute to postnatal growth. The satellite cell population is established during fetal muscle development, through the retention of Pax7-expressing myogenic progenitor cells. We hypothesized that realimentation during late gestation would ameliorate the negative effect of poor maternal nutrition during mid-gestation on the fetal myogenic progenitor cell population. To test this hypothesis, 47 ewes pregnant with singletons were fed a control diet of 100% of National Research Council (NRC) requirements (CON) starting at day 25 of gestation. At day 50 of gestation, six ewes were euthanized and the remainder were randomly assigned to one of two diets: CON or 60% of CON (RES). On day 90 of gestation, a subset of ewes were euthanized (n = 7 per treatment) and fetal semitendinosus samples were collected. The remaining ewes were maintained on the current diet (CON-CON, RES-RES) or switched to the alternative diet (CON-RES, RES-CON). On day 130 of gestation, all ewes were euthanized for fetal sample collection (n = 6–7 per treatment). Fetal semitendinosus was cryosectioned and immunostained for detection of Pax7(+) cells followed by image analysis. Data were analyzed using the MIXED procedure in SAS. Semitendinosus from RES lambs had a greater number of Pax7(+) cells but similar total cell numbers to CON offspring, resulting in a greater percentage of Pax7(+) cells at d90 of gestation (CON: 13.22 ± 0.74%; RES: 16.01 ± 0.74%, P = 0.01). At day 130, there was no difference in the percentage of Pax7(+) cells between dietary treatment groups (CON-CON: 7.88 ± 0.80%; CON-RES: 6.34 ± 0.74%; RES-RES: 7.82 ± 0.74%; RES-CON: 6.87 ± 0.74%; P > 0.17). The percentage of Pax7(+) cells decreased from day 90 to day 130, regardless of dietary treatment (P < 0.0001). In summary, restricted maternal nutrition may delay progenitor cell differentiation at mid-gestation.

Melatonin plays a role as a vasodilator. Vasoactive and angiogenic factors are expressed by placental binucleate cells (BNC) and produce chorionic somatomammotropin (CSH), known to impact fetal and placental growth. We hypothesized that melatonin supplementation and restricted nutrition from mid- to late-gestation would alter CSH concentration and some characteristics of BNC in placenta. At day 50 of gestation, ewes carrying singletons were randomly assigned to a 2 × 2 factorial design and were fed either an adequate (ADQ; 100% NRC; n = 15) or restricted (RES; 60% NRC; n = 15) diet supplemented with 0 (CON, n = 14) or 5 mg of melatonin (MEL; n = 16). Placentomes were collected on day 130 of gestation and preserved in formalin for histological analysis. Cotyledon (COT) were snap frozen for western immunoblotting analyses. Tissue sections were stained using biotinylated Dolichos Biflurus (DBA; a marker of fetal membrane) lectin and fluorescein labeled Texas red-avidin and fluorescein labeled Griffonia Simplifolica (BS) lectin (a marker of BNC). The number, area, and diameter of BNC in COT were determined by image analysis. For immunoblotting, protein was extracted from COT in SDS phosphate buffer, loaded equally, and separated on 12.5% polyacrylamide gels. Protein was transferred to PVDF membranes and incubated with rabbit anti-CSH. Bands were visualized and imaged. Data were analyzed using Proc Mixed procedure of SAS. Melatonin supplementation and restricted nutrition did not affect BNC number, area, or diameter, or CSH protein expression. While we reject our hypothesis that melatonin supplementation and nutrient restriction would alter the CSH concentration and BNC characteristics in COT, we continue to evaluate if the BNC produce angiogenic or vasoactive factors that may influence placental and mammary gland functions in sheep.

The blood–brain barrier regulates the transport of molecules that convey global energetic status to the feeding circuitry within the hypothalamus. Capillaries within the median eminence (ME) and tight junctions between tanycytes lining the third ventricle (3V) are critical components of this barrier. Herein, we tested the hypothesis that altering the plane of nutrition results in the structural reorganization of tanycytes, tight junctions, and capillary structure within the medial basal hypothalamus. Proopiomelanocortin (POMC) neuronal content within the arcuate nucleus of the hypothalamus (ARC) was also assessed to test whether reduced nutritional status improved access of nutrients to the ARC, while decreasing the access of nutrients of overfed animals. Multiparous, nongestating ewes were stratified by weight and randomly assigned to dietary treatments offered for 75 d: 200% of dietary recommendations (overfed), 100% of dietary recommendations (control), or 60% of dietary recommendations (underfed). The number of POMC-expressing neurons within the ARC was increased (P ≤ 0.002) in underfed ewes. Overfeeding increased (P ≤ 0.01) tanycyte cellular process penetration and density compared with control and underfeeding as assessed using vimentin immunostaining. Immunostaining of tight junctions along the wall of the 3V did not differ (P = 0.32) between treatments. No differences were observed in capillary density (P = 0.21) or classification (P ≥ 0.47) within the ME. These results implicate that changes within the satiety center and morphology of tanycytes within the ARC occur as an adaptation to nutrient availability.

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.

Corpus luteum (CL), a transient endocrine gland critical for reproductive cyclicity and pregnancy maintenance, is controlled by numerous regulatory factors. Although LH is widely recognized as the major regulator, other factors may also affect luteal functions. It has been demonstrated that FSH receptors (FSHR) are expressed not only in ovarian follicles but also in other tissues within the reproductive tract, including the CL. To evaluate FSHR expression in nontreated (nonsuperovulated; experiment 1) or FSH-treated (superovulated; experiment 2) sheep fed a control (C; maintenance), excess (O; 2 × C), or restricted (U; 0.6 × C) diet, CL were collected at the early, mid and/or late luteal phases (n = 5-7 per group). Protein and messenger RNA (mRNA) expression of FSHR were detected in the CL from all groups using immunohistochemistry followed by image analysis and quantitative RT-PCR, respectively. Follicle-stimulating hormone receptor was immunolocalized to steroidogenic small and large and nonsteroidogenic luteal cells. In both experiments, FSHR protein expression was not affected by stage of luteal development or diet. In experiment 1, expression of mRNA for all FSHR variants was greater (P0.02 to 0.0003) at the late phase than mid or early luteal phase, and in experiment 2, it was greater (P0.001) at the mid than early luteal phase. Plane of nutrition did not affect FSHR mRNA expression. Comparison of FSH-treated with nontreated ewes demonstrated that FSH increased FSHR protein expression by 1.5- to 2-fold (P0.0001) in all groups, and mRNA expression by 7- to 30-fold (P0.001) for (1) FSHR-1 in all groups except U at the early luteal phase, (2) FSHR-2 in C, O, and U at the mid-phase, but not early luteal phase, and (3) FSHR-3 in U at the mid-luteal phase. Our data demonstrate that (1) FSHRs are expressed in ovine CL at several stages of luteal development, (2) FSHR protein expression does not change during the luteal phase and is not affected by diet, (3) FSHR mRNA expression not only depends on the stage of the estrous cycle but also not affected by diet in nonsuperovulated or superovulated ewes, and (4) in vivo FSH treatment enhanced FSHR protein and/or mRNA expression in the CL depending on diet and phase of the estrous cycle. Presence of FSHR in the CL indicates a regulatory role of FSH in luteal function in sheep. As very little is known about the possible role of FSH and FSHR in luteal functions, further studies should be undertaken to elucidate the endocrine, molecular, and cellular mechanisms of FSH effects on the CL.

Uteroplacental development is a crucial step facilitating conceptus growth. Normal placental development comprises extensive placental angiogenesis to support fetoplacental transport, meeting the metabolic demands of the fetus. Compromised pregnancies due to maternal stressors such as over or undernutrition, maternal age or parity, altered body mass index, or genetic background result in altered vascular development of the placenta. This negatively affects placental growth and placental function and ultimately results in poor pregnancy outcomes. Nonetheless, the placenta acts as a sensor to the maternal stressors and undergoes modifications, which some have termed placental programming, to ensure healthy development of the conceptus. Sex steroid hormones such as estradiol-17β and progesterone, chemokines such as chemokine ligand 12, and angiogenic/vasoactive factors such as vascular endothelial growth factors, placental growth factor, angiopoietins, and nitric oxide regulate uteroplacental development and hence are often used as therapeutic targets to rescue compromised pregnancies. Interestingly, the presence of sex steroid receptors has been identified in the fetal membranes (developing fetal placenta). Environmental steroid mimetics known as endocrine disrupting compounds disrupt conceptus development and lead to transgenerational impairments by epigenetic modification of placental gene expression, which is another area deserving intense research efforts. This review attempts to summarize current knowledge concerning intrinsic and extrinsic factors affecting selected reproductive functions with the emphasis on placental development.

The aim of this study was to investigate the role of the nitric oxide (NO) system in ovarian function, by determining if arginine (Arg) supplementation impacts follicle number, cell proliferation, and expression of the NO system members in nutritionally compromised ewes. Ewes were randomly assigned into maintenance (C, 100% requirements), excess (O; 2xC), or restricted (U; 0.6xC) diets 8 weeks prior to Arg treatment. Ewes were individually fed twice daily with pelleted diets. Ewes from each nutritional group were randomly assigned to one of two treatments: saline or Arg, which was initiated on day 0 of the estrous cycle and administered 3 times per day. Ovaries were collected at the early-luteal, mid-luteal and late-luteal/follicular phases of the estrous cycle to determine 1) the number of surface follicles, 2) follicle cell proliferation marked by Ki67 protein expression, and 3) expression of endothelial nitric oxide (eNOS; NOS3) and soluble guanylyl cyclase beta (sGC; GUCY1B3) protein and mRNA in granulosa (G) and theca (T) layers using immunohistochemistry followed by image analysis and qPCR, respectively. During nutritional treatment, C maintained body weight, O gained 6±1.2 kg, and U lost 14±1.3 kg. Our data show that: 1) Ki67 was expressed in all ovarian compartments, eNOS protein was detected in blood vessels of T and stroma, and sGC protein was detected in T cells, and blood vessels of T layer and other ovarian compartments; 2) plane of nutrition affected the number of surface follicles, and thus folliculogenesis, cell proliferation in the T layer, eNOS and sGC protein expression in T, and NOS3 and GUCY1B3 mRNA expression in G; 3) Arg treatment affected cell proliferation in G and T, eNOS and sGC protein expression in T, mRNA expression of NOS3 in T in all groups, and GUCY1B3 in G depending on the stage of the estrous cycle; and 4) G and T cell proliferation, and expression of eNOS and sGC protein in T was affected by the stage of the estrous cycle. Our data demonstrated that plane of nutrition and Arg are involved in the regulation of follicular functions in non-pregnant sheep.

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 ANPGT-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 towards 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 PGF2a-mediated vascular destabilization.

Follicle stimulating hormone (FSH) is a well characterized gonadotropin that controls primarily development and functions of ovarian follicles in mammalian species. FSH binds to a specific G protein-coupled receptor (FSHR) belonging to the glycoprotein hormone receptor family that plays an essential role in reproduction. Although the primary location of FSHR is in the gonads (mainly in ovarian follicles), FSHR protein and/or mRNA have also been detected in extragonadal female reproductive tissues including embryo, placenta, endometrium, cervix, ovarian cancer tissues, and/or endometriotic lesions in several species. To determine the pattern of FSHR expression in the uterus and placenta, uterine tissues were collected at the early, mid- and/or late luteal phases of the estrous cycle from non-treated or FSH-treated ewes, and utero-placental tissues were collected during early pregnancy followed by immunohistochemistry and image generation. FSHR was immunolocalized to several uterine and utero-placental compartments including luminal epithelium, endometrial glands and surrounding stroma, myometrium, and endothelium and vascular smooth muscle cells in endometrium, myometrium and mesometrium. Intensity of staining and distribution of FSHR in selected compartments differed and seems to depend on the stage of the estrous cycle or pregnancy, and FSH-treatment. These novel data demonstrate differential expression of FSHR protein indicating that FSH plays a specific role in regulation of uterine and utero-placenta functions in sheep.

Sex steroid hormones are major regulators of uterine and placental growth and functions, as well as many other biological processes. To examine the mRNA expression of nuclear estrogen (ESR1 and 2) and progesterone (PGRAB and B) receptors in different compartments of the uterus and placenta, tissues were collected in experiment 1 on days 16, 20, and 28 after natural mating (NAT) and on day 10 after estrus (nonpregnant controls [NP]); and in experiment 2 on day 22 of NAT, and pregnancies established after transfer of embryos generated through mating of FSH-treated ewes (NAT-ET), in vitro fertilization (IVF), or in vitro activation (parthenotes). In experiment 1, ESR1 expression in endometrial stroma (ES), endometrial glands (EGs), and myometrial blood vessels (MBVs), ESR2 in endometrial blood vessels (EBV), PGRAB in ES, and PGRB in ES, EG, and MBV was greater in pregnant than NP ewes depending on the day of pregnancy. The day of pregnancy affected the expression of ESR1 in MBV, ESR2 in EBV and MBV, and PGRAB in ES. In experiment 2, ESR1, PGRAB, and PGRB in EG, but not in other compartments, was greater in NAT-ET than NAT, and PGRB was greater for NAT-ET than IVF. These data demonstrate that ESR and PGR expression differ in pregnant versus NP ewes in selected compartments and was affected by pregnancy stage or embryo origin in selected utero-placental compartments. Thus, sex steroid hormone mRNA expression is differentially regulated in a spatiotemporal manner in the uterus and placenta and is affected by the application of assisted reproductive technology in sheep.

Inadequate maternal nutrition during gestation negatively impacts offspring muscle development, which may be attenuated by re-alimentation. We hypothesized that restricted maternal nutrition would alter fetal muscle fiber cross-sectional area (CSA) and the percent of Pax7(+) progenitor cells. To test this, 48 primiparous ewes, pregnant with singletons, were fed 100% of NRC requirements (CON) between days 25 and 50 of gestation. At day 50 of gestation, seven ewes were euthanized for fetal sample collection. The remaining ewes were fed either CON or 60% NRC requirements (RES). At day 90 of gestation, seven ewes per diet were euthanized for fetal sample collection. The remaining ewes were maintained on their current diet (CON-CON, RES-RES) or switched to the alternate diet (CON-RES, RES-CON). At day 130 of gestation, all ewes (n = 6 or 7 ewes/diet) were euthanized, and fetal samples were collected. On days 90 and 130, fetal longissimus (LM) and triceps brachii (TB) were collected and immunostained for enumeration of Pax7(+) cells and muscle fiber CSA. The percent of Pax7(+) cells was determined by dividing the number of Pax7(+) cells by the total number of cells. At day 90 of gestation, the percent of Pax7(+) cells was less (P < 0.01) in the TB of RES compared with CON. At day 130, fiber CSA was greater (P < 0.04) in RES-CON compared with CON-CON, CON-RES, and RES-CON in the TB, and was greater (P < 0.02) in RES-CON compared with RES-RES and CON-RES in the LM. An effect of maternal nutrition was not observed for the percent of Pax7(+) cells in the LM, or for CSA at day 90 in either muscle. Thus, restricted maternal nutrition during early to mid-gestation alters the percent of Pax7(+) cells at mid-gestation in a muscle specific manner. Re-alimentation during late gestation increased fiber CSA, demonstrating that re-alimentation alters fetal muscle development.

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.

Sex steroids are important regulators of angiogenesis and growth in reproductive tissues, including the placenta. In experiment (exp.) 1, to examine the expression of a suite of sex steroid receptors throughout early pregnancy, maternal (caruncular [CAR]) and fetal (fetal membranes [FM]) placental tissues were collected on days 14 to 30 after mating and on day 10 after estrus (nonpregnant controls). In exp. 2, to examine the hypothesis that assisted reproductive technology would affect the expression of the same suite of sex steroid receptors, pregnancies were achieved through natural mating (NAT) or transfer of embryos from natural mating (NAT-ET), in vitro fertilization (IVF), or in vitro activation (IVA), and CAR and FM were collected on day 22. In exp. 1, for CAR messenger RNA (mRNA) expression of estrogen receptors (ESR) 1 and 2, nuclear (n) progesterone receptors (PGR) and membrane (m) PGRα, β, and γ were affected (P < 0.02) by pregnancy stage, as were ESR1, nPGR, and mPGRα, β, and γ for FM (P < 0.03). In exp. 2, for CAR, mRNA expression of ESR1 and nPGR was decreased (P < 0.001) in NAT-ET, IVF, and IVA groups compared with NAT. For FM, mRNA expression of ESR1 tended to be greater (P = 0.10) in the IVA group compared with NAT and NAT-ET, and GPER1 was greater (P < 0.05) in NAT-ET and IVF compared with NAT. These data establish the normal pattern of sex steroid receptor mRNA expression in maternal and fetal placenta during early pregnancy in sheep, and in addition, suggest that altered expression of placental sex steroid receptors may be an early event leading to poor placental vascularization and growth after assisted reproductive technology.

OBJECTIVE To determine whether a new catheter design with a low-profile, open-ended rounded rather than pointed Foley catheter tip can reduce mucosal damage to the bladder of ewes. METHODS AND MATERIALS Six ewes were randomly assigned to 1 of 2 indwelling urinary catheters-a 16 Fr Foley catheter or a 16 Fr open-tip CystoSure catheter. After 96 hours, all the animals were sacrificed and their bladder and urethra harvested for analysis. RESULTS Image analysis of the bladder surfaces demonstrated a significant decrease in the percentage of bladder area covered by ulceration and inflammation in sheep with CystoSure catheters compared with Foley catheters (P

The aim of this study was to determine the effects of maternal diet with adequate (A) or high (H) selenium (Se) supplementation on ovarian and uterine characteristics, and onset of puberty in adolescent offspring. Sheep were fed a maintenance (M) diet with ASe or HSe levels from breeding to parturition. From Day 50 to parturition, a portion of the ewes from ASe and HSe groups was fed restricted (R, 60% of M) or excess (E, 140% of M) diet. Immediately after birth, lambs were separated from their dams and given artificial colostrum for 20 hours, followed by milk replacer. From Day 57.3 ± 0.6, ewe lambs were fed a pelleted grower diet until Day 116.3 ± 0.6 when they were transitioned to a finisher diet. From Day 99 to 180, serum samples were collected weekly from jugular vein for progesterone analysis to determine onset of puberty. Reproductive tissues were collected on Day 180.1 ± 0.4 of age. Maternal diet or Se supplementation did not affect uterine or ovarian weight and onset of puberty. However, area under the curve for progesterone was greater (P = 0.05) in ASe compared with HSe groups, and was greater in ASeM than HSeM group. In CLs, labeling index (LI; a proportion of proliferating cells) was less (P < 0.04) in HSeM than ASeM group, and in stroma was less (P < 0.05) in R and E groups than M group. Maternal diet did not affect the LI of any follicle types. For all groups combined, LI was the greatest (P < 0.001) in antral, less in early antral and secondary, and the least in atretic follicles. Our results demonstrate that maternal diet influenced ovarian but not uterine characteristics or onset of puberty. These results indicate that maternal plane of nutrition and/or Se supplementation may have specific effects on reproductive function in offspring.

The duration and timing of inadequate maternal nutrition can have detrimental effects on metabolism and organogenesis in the offspring. Re-alimentation, a common management practice that involves feeding full nutrient requirements following a period of nutrient restriction, may reduce the negative impacts of maternal nutrient restriction. To determine the effects of maternal nutrient restriction and re-alimentation on offspring growth,48 primiparous ewes, confirmed pregnant with singletons, were fed a control diet consisting of100% NRC requirements (CON) starting on day25 of gestation. On day50 of gestation, ewes (n = 7) were euthanized and fetal liver, muscle, and blood samples were collected. The remaining animals were fed either CON or60% NRC requirements (RES). On day90 of gestation, a portion of ewes were euthanized (n = 7 per treatment) and fetal samples and weights were collected. Remaining ewes were maintained on the current diet (CON-CON, n = 6; RES-RES, n = 7) or switched to the alternative diet (CON-RES, RES-CON; n = 7/treatment). On day130 of gestation, all remaining ewes were euthanized. All fetal BW, liver, longissimus muscle, semitendinosus, and triceps brachii weights were determined for each day of gestation. Fetal BW’s were not different between treatment groups (P = 0.29; P = 0.83). Fetal liver weights decreased12.89% in RES-RES compared with CON-CON at day130 (P = 0.049), but were not different at day90 (P = 0.69). There was a tendency for decreased semitendinosus weight in RES group compared with CON at day90 (P = 0.055). Liver lipid droplet accumulation was analyzed for day90 and130 using histochemistry and an effect of maternal nutrition was not observed (P = 0.562). In summary, maternal nutrient restriction reduces offspring muscle and liver growth. To gain insight into the effects of maternal nutrient restriction and re-alimentation on liver development and metabolism, analysis of liver morphology, gene expression, and global metabolomics are needed. Supported by USDA-AFRI grant2016-67016-24884

The origin of embryos including those created through assisted reproductive technologies might have profound effects on placental and fetal development, possibly leading to compromised pregnancies associated with poor placental development. To determine the effects of embryo origin on fetal size, and maternal and fetal placental cellular proliferation and global methylation, pregnancies were achieved through natural mating (NAT), or transfer of embryos generated through in vivo (NAT-ET), IVF, or in vitro activation (IVA). On Day 22 of pregnancy, fetuses were measured and placental tissues were collected to immunologically detect Ki67 (a marker of proliferating cells) and 5-methyl cytosine followed by image analysis, and determine mRNA expression for three DNA methyltransferases. Fetal length and labeling index (proportion of proliferating cells) in maternal caruncles (maternal placenta) and fetal membranes (fetal placenta) were less (P < 0.001) in NAT-ET, IVF, and IVA than in NAT. In fetal membranes, expression of 5-methyl cytosine was greater (P < 0.02) in IVF and IVA than in NAT. In maternal caruncles, mRNA expression for DNMT1 was greater (P < 0.01) in IVA compared with the other groups, but DNMT3A expression was less (P < 0.04) in NAT-ET and IVA than in NAT. In fetal membranes, expression of mRNA for DNMT3A was greater (P < 0.01) in IVA compared with the other groups, and was similar in NAT, NAT-ET, and IVF groups. Thus, embryo origin might have specific effects on growth and function of ovine uteroplacental and fetal tissues through regulation of tissue growth, DNA methylation, and likely other mechanisms. These data provide a foundation for determining expression of specific factors regulating placental and fetal tissue growth and function in normal and compromised pregnancies, including those achieved with assisted reproductive technologies.

To determine the effect of feed intake and arginine treatment during different stages of the estrous cycle on pancreatic mass, digestive enzyme activity, and histological measurements, ewes (n = 120) were randomly allocated to 1 of 3 dietary groups; control (CON; 2.14-Mcal metabolizable energy/kg), underfed (UF; 0.6 × CON), or overfed (OF; 2 × CON) over 2 yr. Estrus was synchronized using a controlled internal drug release device for 14 d. At controlled internal drug release withdrawal, ewes from each dietary group were assigned to 1 of 2 treatments; Arg (L-Arg HCl, 155-μmol/kg BW) or Sal (approximately 10-mL saline). Treatments were administered 3 times daily via jugular catheter and continued until slaughter on d (day) 5 and 10 of the second estrus cycle (early luteal phase, n = 41 and mid-luteal phase, n = 39; yr 1) and d 15 of the first estrus cycle (late luteal phase, n = 40; yr 2). A blood sample collected from jugular catheters for serum insulin analysis before slaughter. The pancreas was then removed, trimmed of mesentery and fat, weighed, and a sample snap-frozen until enzyme analysis. Additional pancreatic samples were fixed in 10% formalin solution for histological examination of size and distribution of insulin-containing cell clusters. Data were analyzed as a completely randomized design with a factorial arrangement of treatments. Diet, treatment, and diet × treatment were blocked by yr and included in the model with initial BW used as a covariate. Day of the estrous cycle was initially included in the model but later removed as no effects (P > 0.10) were observed for any pancreatic variables tested. Overfed ewes had the greatest (P < 0.001) change in BW, final BW, change in BCS, and final BCS. A diet × treatment interaction was observed for change in BW and final BW (P ≤ 0.004). Overfed and CON had increased (P < 0.001) pancreas weight (g) compared with UF ewes. Protein concentration (g/pancreas) was the lowest (P < 0.001) in UF ewes, whereas protein content (mg/kg BW) was greater (P = 0.03) in UF than OF ewes. Activity of α-amylase (U/g, kU/pancreas, U/kg of BW, and U/g protein) and trypsin (U/pancreas) was greater (P ≤ 0.003) in OF than UF ewes. Serum insulin was the greatest (P < 0.001) in OF ewes. No effects were observed for pancreatic insulin-containing cell clusters. This study demonstrated that plane of nutrition affected several measurements of pancreatic function; however, the dosage of Arg used did not influence pancreatic function.

The aim of this study was to evaluate lipid droplet (LD) expression in uteri of FSH-treated or nontreated sheep administered with arginine (Arg) or vehicle (saline, Sal) and fed a control (C), excess (overfed, O) or restricted (underfed, U) diet. In experiment 1, ewes from each diet were randomly assigned to Arg or Sal treatments administered three times daily starting on Day 0 of the first estrous cycle until blood sample and uterine tissue collection at the early- or mid-luteal phase of the second estrous cycle or the late-luteal phase of the first estrous cycle. In experiment 2, ewes were injected twice daily with FSH on Days 13 to 15 of the first estrous cycle, and blood samples and uterine tissue were collected at the early- and mid-luteal phases of the second estrous cycle. Cryopreserved in optimum cutting temperature (OCT) compound, cross-sections of uterine horn were stained with boron-dipyrromethene (BODIPY; marker of LDs) followed by 4′,6-diamidino-2-phenylindole (DAPI) staining and image analysis to determine the proportion (%) of area occupied by LD in luminal epithelium (LE) and endometrial glands (EGs). Control ewes maintained, O ewes gained, and U ewes lost body weight during the experiments. Serum progesterone concentration was not affected by nutritional plane or Arg treatment and was 5.5-fold greater in FSH- than Sal-treated ewes. LDs were detected in LE and superficial EG (close to LE) but not deep EG, or other uterine compartments, and area occupied by LD was greater in LE than in EG. In experiment 1, in LE and EG, area occupied by LDs was greater in C than in O or U; greater in Arg than in Sal; and greater at the late-, less at mid-, and least at early-luteal phases. In experiment 2, in LE and EG, area occupied by LDs was greater at mid- than in early-luteal phase. Comparison of data from FSH-treated and nontreated ewes (e.g., experiment 1 vs. experiment 2) demonstrated that FSH increased the area occupied by LD in LE and EG regardless of diet. Interactions between FSH treatment, stage of the estrous cycle, and plane of nutrition demonstrated that FSH increased the area occupied by LD in LE and EG at the mid-luteal phase in O and U. Thus, LDs are differentially distributed in uterine compartments, and area occupied by LD in endometrium is affected by nutritional plane, Arg or FSH, and stage of the estrous cycle. Such changes in dynamics of LD in the endometrium during the estrous cycle indicate their specific role in uterine functions.

Gap junctions play a major role in direct, contact-dependent cell-cell communication, and they have been implicated in the regulation of cellular metabolism and the coordination of cellular functions during growth and differentiation of organs and tissues. Gap junctional channels, composed of connexin (Cx) proteins, have been detected and shown to be influenced by hormones (eg, estrogen and progesterone) in uterine and placental tissues in several species. We hypothesized that (1) the messenger RNA (mRNA) for Cx26, Cx32, Cx37, and Cx43 is expressed in the uterus of ovariectomized sheep treated with estradiol-17β (E2) and in ovine placenta during early pregnancy, (2) E2-treatment of ovariectomized ewes would cause time-specific changes in Cx26, Cx32, Cx37, and Cx43 mRNA expression (experiment 1), and (3) expression of these 4 Cx would vary across the days of early pregnancy (experiment 2) and will be affected by embryo origin (ie, after application of assisted reproductive technologies [ARTs]; experiment 3). Thus, we collected uterine tissues at 0 to 24 h after E2 treatments (experiment 1), and placental tissues during days 14 to 30 of early pregnancy after natural (NAT) breeding (experiment 2) and on day 22 of early pregnancy established after transfer of embryos generated through natural breeding (NAT-ET), in vitro fertilization (IVF), or in vitro activation (IVA, parthenotes; experiment 3). In experiment 1, the expression of Cx26, Cx37, and Cx43 mRNA increased (P < 0.05) and Cx32 mRNA decreased (P < 0.06) in both caruncular and intercaruncular tissues after E2 treatment. In experiment 2, during early pregnancy, there were significant changes (P < 0.01) across days in the expression of Cx26, Cx37, and Cx43 mRNA in the maternal placenta, accompanied by changes (P < 0.001) in Cx37 and Cx43 mRNA in the fetal placenta. In experiment 3, in maternal placenta, Cx32 mRNA expression was decreased (P < 0.001) in NAT-ET, IVF, and IVA groups compared to the NAT group; but in fetal placenta, Cx32 mRNA expression was increased (P < 0.05) in NAT-ET, IVF and IVF groups, and Cx26 mRNA expression was increased (P < 0.05) in IVA compared to NAT group. These data suggest that Cx26, Cx32, Cx37, and Cx43 play specific roles in E2-regulated uterine function and in placental development during early gestation both after natural mating and with application of ART.

The aim was to localize chemokine ligand twelve (CXCL12) in sheep placental tissues during early gestation and after assisted reproductive technologies (ART). Uteri were collected from naturally (NAT) mated ewes and ewes receiving embryo transfer (ET), in vitro fertilization (IVF) or in vitro activation (IVA). CXCL12 was immunolocalized to endometrial stroma, glands, and trophoblast. Greater CXCL12 immunoreactivity was present in trophoblast on day 22 and 24 and in NAT ewes compared to IVF and IVA. Increased CXCL12 expression suggests CXCL12 promotes implantation and placentation. Decreased CXCL12 in IVF and IVA embryos, may compromise pregnancy establishment when utilizing ART methods.

The purpose of this retrospective study was to establish a prognosis for implantation, pregnancy and live birth rates in stimulated IVF cycles after transferring embryos derived from: 1/ retrieved immature oocytes that matured overnight in vitro (late mature group: LM); 2/ retrieved immature oocytes that matured overnight in vitro and were added to the embryos derived from retrieved mature oocytes (mixed embryos group: MX); and 3/ retrieved mature oocytes (mature group: M). The obtained implantation, clinical pregnancy and live birth rates for the LM group were: 5.6%, 11.4%, 11.4%; for the MX group were: 4.2%, 14.6%, 11.6%; and for the M group were: 14.6%, 45.2% and 33.3%, respectively. These measurements were significantly lower p0.05 for the LM and MX groups in comparison to the M group. The number of oocytes retrieved and the number of embryos transferred were the lowest (p0.001-0.05) for the LM group. It is concluded, that the retrieved immature oocytes are able to mature during overnight culture in vitro, be fertilized and provide developmentally competent embryos with the prognosis of 11% for the successful delivery.

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.

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, and3b; 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 forDNMT3bdecreased, but mRNA forDNMT1and3aincreased. 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 receptorsFLT1andKDR; angiopoietins 1 and 2 and their receptorTEK; endothelial nitric oxide synthase and the NO receptorGUCY13B; 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.

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 (PPANGPT) 1 and 2, ANGPT receptorTEK, endothelial nitric oxide synthase, and hypoxia-inducible factor 1α increased (PP

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 examine the effects of maternal supranutritional selenium (Se) and nutrient restriction during mid and late gestation on placental characteristics and fetal liver glycogen, ewes received either adequate Se (ASe) or high Se (HSe) prior to breeding. On d 64 of gestation, ASe and HSe ewes remained at 100% of requirements (controls; CON) or were restricted (RES; 60% of requirements). On d 135 of gestation, fetal weight (P ≤ 0.08) was greatest in both HSe and CON ewes. Placentome number, mass, and caruncular and cotyledonary weight were not different (P ≥ 0.17) among treatments. Fetal mass:placental mass ratio was less (P = 0.06) in RES compared to CON ewes. Compared to ASe, HSe exhibited increased (P ≤ 0.08) cellular proliferation and DNA concentration and decreased (P = 0.07) cellular size in cotyledonary tissue. Nutritional restriction decreased (P ≤ 0.08) cotyledonary protein concentration and cellular size. VEGF receptor 1 (Flt) mRNA in cotyledonary tissue was greater in HSe compared with ASe ewes (P = 0.06) and in RES compared with CON ewes (P = 0.08). There was no effect of diet on caruncular growth variables (P ≥ 0.13) or on placental vascularity (P ≥ 0.11). Progesterone was greater (P ≤ 0.08) in ASe–RES ewes compared to all groups at d 90 and ASe–CON and HSe–CON at d 104. Although fetal glucose and cortisol concentrations were not affected by diet, fetal liver glycogen was greater (P = 0.04) in ASe–RES compared to ASe–CON and HSe–RES ewes with HSe–CON being intermediate. Both Se and nutritional plane may impact placental function and fetal growth, as fetal weight and liver glycogen are altered despite similar placental vascularity measurements.

For singleton, twin, and triplet pregnancies, uteri were collected on day 140 of pregnancy. For each ewe (n = 18), placentomes were fixed by arterial perfusion supplying the fetal (cotyledon) and maternal placenta (caruncle). Tissue sections were stained for determination of vascularity by image analysis. Further, protein expression for factor VIII, vascular endothelial growth factor (VEGF) and its receptor, VEGFR1, as well as basic fibroblast growth factor (FGF2) and its receptor, FGFR, in tissue sections was determined by immunohistochemistry and image analyses. Cotyledonary and caruncular samples were analyzed for expression of mRNA for Vegf and its two receptors, Vegfr1 and Vegfr2, as well as Fgf2 and Fgfr. Fetal number did not affect placental capillary density or factor VIII expression, whereas increased fetal number reduced total cotyledon and caruncle capillary volume. While expression of Vegf, Vegfr1, Vegfr2, and Fgfr mRNA in cotyledonary but not caruncular tissue was greater in twin pregnancies compared to singleton and triplet pregnancies, protein expression of VEGF in the placentome decreased with increasing numbers of fetuses, VEGFR1 did not change, and FGFR was greater in twin versus singleton and triplet pregnancies. Fetal number did not affect the expression of Fgf2 mRNA in placental tissues, whereas FGF2 protein expression was less in triplet compared to singleton and twin pregnancies. Reduced fetal and placental weights in twins and/or triplet pregnancies are associated with an overall decrease in total placental vascularity, VEGF and FGF2 and/or FGFR protein expression, but not in angiogenic factor mRNA expression or VEGFR1 protein expression in sheep.

Accumulation of lipid droplets (LD) in luteal cells likely is important for energy storage and steroidogenesis in the highly metabolically active corpus luteum (CL). The objective of this study was to determine the effect of plane of nutrition on progesterone (P4) secretion, and lipid droplet number and size in cultured ovine luteal cells. Ewes were randomly assigned to one of three nutritional groups: control (C; 100% NRC requirements, n=9), overfed (O; 2×C, n=12), or underfed (U; 0.6×C, n=10). Superovulation was induced by follicle stimulating hormone injections. At the early and mid-luteal phases of the estrous cycle, CL were dissected from ovaries, and luteal cells isolated enzymatically. Luteal cells were incubated overnight in medium containing serum in chamber slides. Media were then changed to serum-free and after 24h incubation, media were collected for P4 analysis, and cells were fixed in formalin and stained with BODIPY followed by DAPI staining. Z-stacks of optical sections of large and small luteal cells (LLC and SLC, respectively) were obtained using a laser-scanning microscope. Rendered 3D images of individual LLC and SLC were analyzed for cell volume, and total and individual LD volume, number and percentage of cellular volume occupied by LD by using Imaris software. Concentrations of P4 in serum and media were greater (P