Eyal Klipper

Regulation of Angiogenesis-Related Prostaglandin F2alpha-Induced Genes in the Bovine Corpus Luteum

ABSTRACT We recently compared prostaglandin F2alpha (PG)-induced global gene expression profiles in PG-refractory, bovine corpus luteum (CL) collected on Day 4 of the estrous cycle, versus PG-responsive, Day 11 CL. Transcriptome analyses led us to study the regulation of angiogenesis-related genes by PG and their functions in luteal endothelial cells (ECs). We found that PG regulated angiogenesis-modulating factors in a luteal stage-dependent way. A robust increase in FGF2 expression (mRNA and protein) occurred in the PG-refractory Day 4 CL promoting CL survival and function. Inhibitors of FGF2 action, thrombospondin 1 and 2, their receptor (CD36), and PTX3 were upregulated by PG specifically in Day 11 CL undergoing luteolysis. VEGF mRNA decreased 4 h post-PG in both Day 4 and Day 11 CL. The resulting destabilization of blood vessels in Day 11 CL is expected to weaken the gland and reduce its hormonal output. These genes were expressed in dispersed luteal ECs and steroidogenic cells; however, thrombospondin 1 and FGF2 were more abundant in luteal ECs. Expression of such genes and their ability to modulate FGF2 actions were investigated. Similar to its in vivo effect, PG, in vitro, stimulated the expression of thrombospondins and PTX3 genes in several luteal cell models. Importantly, these factors influenced the angiogenic properties of luteal ECs. FGF2 dose-dependently enhanced cell migration and proliferation, whereas thrombospondin 1 and PTX3 inhibited FGF2 actions in luteal ECs. Collectively, the data presented here suggest that, by tilting the balance between pro- and antiangiogenic factors, PG can potentially control the ability of the CL to resist or advance toward luteolysis.

Induction of Endothelin-2 Expression by Luteinizing Hormone and Hypoxia: Possible Role in Bovine Corpus Luteum Formation

The pattern and regulation of endothlin-2 (EDN2) expression and its putative roles in bovine ovaries were investigated. EDN2 mRNA was determined in corpus luteum (CL) and during folliculoluteal transition induced by GnRH in vivo. EDN2 was elevated only in the early CL and was not present in older CL. In the young CL, EDN2 mRNA was identified mainly in luteal cells but not endothelial cells that expressed the EDN1 gene. Similarly, in preovulatory follicles, EDN2 was expressed in the granulosa cells (GCs) and not in the vascular theca interna. LH and hypoxia are two major stimulants of CL formation. Therefore, GCs were cultured with bovine LH, under hypoxic conditions. GCs incubated with bovine LH resulted in increased EDN2 mRNA 42 h later. CoCl2, a hypoxia-mimicking agent, elevated EDN2 in GCs in a dose-dependent manner. Incubation of the human GC line (Simian virus 40 large T antigen) under low oxygen tension (1%) augmented EDN2 6 and 24 h later. In these two cell types, along with EDN2, hypoxia augmented VEGF. EDN2 induced in GCs changes that characterize the developing CL: cell proliferation as well as up-regulation of vascular endothelial growth factor and cyclooxygenase-2 (mRNA and protein levels). Human chorionic gonadotropin also up-regulated these two genes. Small interfering RNA targeting EDN-converting enzyme-1 effectively reduced its mRNA levels. This treatment, expected to lower the mature EDN2 peptide production, inhibited VEGF mRNA levels and GC numbers. Together these data suggest that elevated EDN2 in the early bovine CL, triggered by LH surge and hypoxia, may facilitate CL formation by promoting angiogenesis, cell proliferation, and differentiation.

Immune responses of chickens to dietary protein antigens. I. Induction of systemic and intestinal immune responses following oral administration of soluble proteins in the absence of adjuvant.

Oral administration of protein antigens in solution leads to the development of oral tolerance in most mammals but rarely so in the chicken. As dietary proteins are not expected to be immunogenic, the present study was undertaken to evaluate immunological consequences following oral exposure to protein antigens in chicks, and to determine whether or not this form of antigen is ignored. Chicks and turkey poults were fed solutions containing bovine serum albumin (BSA), porcine serum albumin, beta-lactoglobulin or bovine hemoglobin over a period of 6 days (25mg/chick/day). At different time points after feeding serum and bile were examined for presence of specific antibodies by ELISA. Surprisingly, the fed antigens induced robust antibody responses in the absence of added adjuvant. This immune response was further characterised to show that (1) a daily feeding regimen was more immunogenic than single dose feedings, (2) by using a daily feeding regimen, as little as 2mg/chick/day was fully immunogenic, (3) effective immunization was attained in chicks older than 10 day of age, (4) the main antibody class in the serum was IgG, and (5) high IgA levels were detected in the bile after booster feedings. These observations are difficult to reconcile with current concepts on peripheral tolerance to innocuous antigens, and indicate that the bird regulates tolerance and response in a manner different from that described in mammals.

Subclinical, chronic intramammary infection lowers steroid concentrations and gene expression in bovine preovulatory follicles

Chronic, subclinical intramammary infection depresses fertility. We previously found that 30% of subclinical mastitic cows exhibit delayed ovulation, low circulating estradiol levels, and delayed luteinizing hormone surge. We examined the function of preovulatory follicles of cows experiencing subclinical mastitis or a past event of acute clinical mastitis. Cows were diagnosed for mastitis by somatic cell count and bacteriological examination. All clinical infections were caused by Escherichia coli, and most subclinical infections were caused by Streptococcus dysgalactiae and coagulase-negative staphylococci. On day 6 of the cycle, cows received PGF2α; 42 h later, follicular fluids and granulosa cells or theca cells were aspirated from preovulatory follicles in vivo or following slaughter, respectively. Overall, follicular estradiol and androstenedione concentrations in the subclinical group (n = 28) were 40% lower (P < 0.05) than those in uninfected cows (n = 24) and lower than in past clinical mastitic cows (n = 9). Distribution analysis revealed a clear divergence among subclinical cows: one-third (9/28) exhibited low follicular estradiol; the other two-thirds had normal levels similar to all uninfected (P < 0.01) and most clinical cows (P < 0.08) that had normal follicular estradiol levels. Subclinical normal-estradiol cows had twofold higher (P < 0.05) circulating estradiol concentrations and sevenfold and fourfold higher (P < 0.05) follicular androstenedione levels and estradiol-to-progesterone ratio, respectively, than subclinical low-estradiol cows. Follicular progesterone level was not affected. Reduced expression (P < 0.05) of LHCGR in theca and granulosa cells, CYP11A1 (mRNA and protein) and CYP17A1 in theca cells, and CYP19A1 in granulosa cells may have contributed to the lower follicular steroid production in the subclinical low-estradiol subgroup. StAR and HSD3B1 in theca cells and FSHR in granulosa cells were not affected. Mastitis did not alter follicular growth dynamics, and no carryover effect of past clinical mastitis on follicular function was detected. These data indicate that a considerable proportion (one-third) of subclinical mastitic cows have abnormal follicular steroidogenesis, which can explain the reproductive failure associated with this disease.

Small Interfering RNA Molecules Targeting Endothelin-Converting Enzyme-1 Inhibit Endothelin-1 Synthesis and the Invasive Phenotype of Ovarian Carcinoma Cells

Endothelin-1 (ET-1) has been implicated in the progression of various cancers, including ovarian carcinoma. We found that the ovarian carcinoma cell lines ES2 and OVCAR3 and tumors from different anatomic sites expressed ET-1 system members [ET receptor A and ET-converting enzyme-1 (ECE-1)]. However, only ECE-1 was significantly higher in the solid tumors compared with effusions. We therefore investigated the effect of RNA interference-induced knockdown of ECE-1, the key enzyme in ET-1 production, on these two ovarian carcinoma cell lines. Small interfering RNA (siRNA) targeting of ECE-1 markedly reduced ECE-1 mRNA and protein levels, which subsequently led to 80% to 90% inhibition of ET-1 peptide secretion by the cells. ECE-1 silencing also profoundly affected the behavior of tumor cells compared with cells treated with scrambled siRNA. Silenced cells exhibited (a) reduced ET-1-dependent p44/42 mitogen-activated protein kinase phosphorylation; (b) decreased invasiveness and matrix metalloproteinase-2 activity; (c) improved adhesion to basal lamina proteins, laminin-1, and collagen IV; and (d) increased E-cadherin, an epithelial adhesion molecule, and reduced N-cadherin expression, a mesenchymal marker. Altered cell adherence is one of the hallmarks of the transformed phenotype, often characterized by the loss of the epithelial features and the gain of a mesenchymal phenotype. ECE-1 ablation did not, however, alter viable ovarian carcinoma cell numbers. Addition of exogenous ET-1 reversed the effects cited above. Taken together, these data indicate that siRNA is an effective tool for manipulating ECE-1 expression, ET-1 biosynthesis, and invasiveness of ovarian carcinoma. ECE-1 silencing may therefore develop into a promising novel anticancer therapy.

The effect of chronic feeding of diacetoxyscirpenol and T-2 toxin on performance, health, small intestinal physiology and antibody production in turkey poults

1. The effects of feeding T-2 toxin or diacetoxyscirpenol (DAS) at levels up to 1 ppm for 32 d on performance, health, small intestinal physiology and immune response to enteral and parenteral immunisation were examined in young poults. 2. Slight improvement in growth was observed in some groups of poults fed T-2 or DAS mycotoxins for 32 d, with no change in feed efficiency. Feeding both T-2 and DAS resulted in oral lesions which had maximal severity after 7-15 d. 3. Mild intestinal changes were observed at 32 d but no pathological or histopathological lesions were found. Both mycotoxins altered small intestinal morphology, especially in the jejunum where villi were shorter and thinner. In addition, both DAS and T-2 mycotoxins enhanced the proportion of proliferating cells both in the crypts and along the villi. Migration rates were reduced in the jejunum of poults fed T-2 toxin but did not change in the duodenum or in poults fed DAS. 4. No significant effects of T-2 or DAS were observed on antibody production to antigens administered by enteral or parenteral routes. 5. This study indicates that tricothecene toxins at concentrations of up to 1 ppm for more than 30 d influenced small intestinal morphology but did not affect growth or antibody production.

Immediate and carryover effects of Gram-negative and Gram-positive toxin-induced mastitis on follicular function in dairy cows

This study compared immediate and carryover effects of mastitis induced by Gram-negative endotoxin (E. coli LPS) and Gram-positive exosecretions (Staph. aureus ex.) on preovulatory follicle function. Synchronized, uninfected cyclic lactating Holstein cows were treated with PGF(2α) on day 6 of the cycle and 36 h later, a dose of either E. coli LPS (n = 8), S. aureus ex. (n = 10), or saline (n = 9) was administered into the mammary gland. Follicular fluids and granulosa cells were aspirated 6 h later from the preovulatory follicles and cows were treated with GnRH. This (cycle 1; immediate effect) was repeated three times (excluding the mammary injections) to induce three 7 d cycles (cycles 2, 3, and 4; carryover effect). E. coli LPS increased body temperature, plasma cortisol concentration, and somatic cell count (SCC), whereas S. aureus ex. induced a minor, subclinical elevation of SCC and slight rise (NS) in body temperature and cortisol concentration. Follicular estradiol, androstenedione, and progesterone concentrations in the E. coli LPS group decreased (P < 0.05) in cycle 1 to about 40%, 13%, and 35%, respectively, of control levels, whereas in the S. aureus ex. group, only estradiol decreased (P < 0.05), to 56% of control concentrations. In cycles 3 and 4, follicular steroids in the E. coli LPS group returned to control concentrations, whereas in the S. aureus ex. group, follicular concentrations of estradiol and androstenedione were lower (P < 0.10) than in controls. In the control group, the concentrations of all follicular and circulating steroids remained stable (P > 0.05) throughout the study. Follicle size was similar in all groups, but the S. aureus ex. treatment caused a decrease (P < 0.02) in the number of follicles developed in cycles 3 and 4. The mRNA expression of steroidogenic genes and LHCGR in the granulosa cells was not affected (P > 0.05) by either treatment during the study, except for a tendency toward lower (P < 0.1) expression in cycle 1 and lower (P < 0.05) expression in cycle 4 of the latter in the S. aureus ex. group. Strain levels, such as SCC and body temperature, following toxin injection correlated well with the magnitude of the immediate decline in follicular steroids. As is typical for Gram-negative clinical events, E. coli LPS-induced acute mastitis caused immediate, short-term, but not long-term impairment of follicular responses, whereas the Gram-positive S. aureus ex.-induced subclinical mastitis exhibited both immediate and carryover disruptive effects on preovulatory follicle function.

Endothelin-converting enzyme-1, abundance of isoforms a-d and identification of a novel alternatively spliced variant lacking a transmembrane domain.

Endothelin-converting enzyme-1 (ECE-1) cleaves big endothelins, as well as bradykinin and β-amyloid peptide. Several isoforms of ECE-1 (a-d) have been identified to date; they differ only in their NH2 terminus but share the catalytic domain located in the COOH-terminal end. Using quantitative PCR, we found ECE-1d to be the most abundant type in several endothelial cells (EC) types. In addition to full-length ECE-1 forms we have identified novel, alternatively spliced mRNAs of ECE-1 b-d. These splice variants (SVs) lack exon 3′, which codes for the transmembrane region and is present in full-length forms. SVs mRNA were highly expressed in EC derived from macro and microvascular beds but much less so in other, non-endothelial cells expressing ECE-1, which suggests that the splicing mechanism is cell-specific. Analyses of ECE-1d and its SV form in stably transfected HEK-293 cells revealed that both proteins were recognized by anti COOH-terminal ECE-1 antibodies, but anti NH2-terminal antibodies only bound ECE-1d. The novel protein, designated ECE-1 sv, has an apparent molecular mass of 75 kDa; by using site-directed mutagenesis its start site was identified in a region common to all ECE-1 forms suggesting that ECE-1 b-d SV mRNAs are translated into the same protein. In agreement with the findings demonstrating common COOH terminus for ECE-1sv and ECE-1d, both exhibited a similar catalytic activity. However, immunofluorescence staining and differential centrifugation revealed a distinct intracellular localization for these two proteins. The presence of ECE-1sv in different cellular compartments than full-length forms of the enzyme may suggest a distinct physiological role for these proteins.

The role of hypoxia-induced genes in ovarian angiogenesis.

The hypoxic microenvironment that occurs in fast-growing tissue such as the corpus luteum (CL) is a major contributor to its ability to survive via the induction of an intricate vascular network. Cellular responses to hypoxia are mediated by hypoxia-inducible factor-1 (HIF-1), an oxygen-regulated transcriptional activator. HIF-1, a heterodimer consisting of a constitutively-expressed β subunit and an oxygen-regulated α subunit, binds to the hypoxia responsive element (HRE) present in the promoter regions of responsive genes. This review summarises evidence for the involvement of hypoxia and HIF-1α in CL development and function. Special emphasis is given to hypoxia-induced, luteal cell-specific expression of multiple genes (vascular endothelial growth factor A (VEGFA), fibroblast growth factor 2 (FGF-2), prokineticin receptor 2 (PK-R2), stanniocalcin 1 (STC-1) and endothelin 2 (EDN-2) that participate in the angiogenic process during CL formation.

Response, tolerance and ignorance following oral exposure to a single dietary protein antigen in Gallus domesticus

The objective of this investigation was to analyze conditions leading to antibody responses against innocuous dietary protein antigens in the chick. The physical form of antigen was found to be important for immunization of mature chicks: bovine serum albumin (BSA) in solution was a powerful immunogen, while BSA powder was ignored. When BSA was fed to newly hatched chicks, either in solution or as powder, specific oral tolerance was induced. Tolerance increased with the dose of antigen fed, and was most effective in suppressing BSA-specific oral immunization. Hence, immune responses of mature chicks to innocuous dietary proteins are not likely to constitute a health hazard due to (a) prevalence of oral tolerance induced at hatch, and (b) availability of dietary proteins in solid form.