Bovine somatotropin attenuates phorbol ester-induced prostaglandin F2alpha production in bovine endometrial cells

The recent observation that bovine somatotropin (bST) treatment at a timed insemination improves pregnancy rates in lactating dairy cows raises the possibility that growth hormone (GH) may modulate the endocrine and biochemical cross talk between the conceptus and maternal uterus at the time of pregnancy establishment in cattle. The objective of this study was to characterize the cellular and molecular mechanisms by which exogenous GH affects phorbol ester-induced prostaglandin F2alpha (PGF2alpha) production in cultured bovine endometrial (BEND) cells. Serum-deprived BEND cells were incubated with or without recombinant bovine GH (rbGH), insulin-like growth factor (IGF)-I, recombinant bovine interferon (rbIFN)-tau or a combination of rbGH + rbIFN-tau for 3 h and then treated with phorbol 12,13-dibutyrate (PDBu) for an additional 6 h. Exogenous PDBu increased PGF2alpha secretion and steady-state levels of COX-2 mRNA within 3 h. Priming of BEND cells with rbGH reduced PGF2alpha response to PDBu, whereas cotreatment with IGF-I amplified PDBu induction of PGF2alpha. Preincubation of cell monolayers with rbIFN-tau suppressed PGF2alpha and COX-2 mRNA responses to PDBu. Inhibitory effects of rbGH and rbIFN-tau on PDBu-induced PGF2alpha production were additive. Results provide the first direct evidence that supplemental bST may interact with conceptus-secreted IFN-tau to modulate PGF2alpha secretion at the critical time of maternal recognition of pregnancy.     

Bacterial infection of endometrial stromal cells influences bovine herpesvirus 4 immediate early gene activation: a new insight into bacterial and viral interaction for uterine disease

Experimental infection with the gamma-herpesvirus bovine herpesvirus 4 (BoHV-4) rarely establishes disease, yet BoHV-4 is commonly associated with uterine disease in cattle. Uterine disease involves co-infection with bacteria such as Escherichia coli, which stimulate the production of prostaglandin E(2) (PGE(2)) by endometrial cells. BoHV-4 replication depends on immediate early 2 (IE2) gene transactivation and, in the present study, PGE(2), E. coli or its lipopolysaccharide upregulated the IE2 gene promoter in uterine cells. Bacterial co-infection is important for BoHV-4 uterine disease.     

Short communication: Pasteurization of milk abolishes bovine herpesvirus 4 infectivity

Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus highly prevalent in the cattle population that has been isolated from the milk and the serum of healthy infected cows. Several studies reported the sensitivity and the permissiveness of some human cells to BoHV-4 infection. Moreover, our recent study demonstrated that some human cells sensitive but not permissive to BoHV-4 support a persistent infection protecting them from tumor necrosis factor-α-induced apoptosis. Together, these observations suggested that BoHV-4 could represent a danger for public health. To evaluate the risk of human infection by BoHV-4 through milk or serum derivatives, we investigated the resistance of BoHV-4 to the mildest thermal treatments usually applied to these products. The results demonstrated that milk pasteurization and thermal decomplementation of serum abolish BoHV-4 infectivity by inactivation of its property to enter permissive cells. Consequently, our results demonstrate that these treatments drastically reduce the risk of human infection by BoHV-4 through treated milk or serum derivatives.     

Control of cyclic AMP concentration in bovine endometrial stromal cells by arachidonic acid

Second messenger signalling through cyclic AMP (cAMP) plays an important role in the response of the endometrium to prostaglandin (PG) E(2) during early pregnancy. Arachidonic acid, which is a by-product of the luteolytic cascade in ruminants, is a potential paracrine signal from the epithelium to the stroma. We investigated the effects of arachidonic acid on the response of the stroma to PGE(2). cAMP was measured in bovine endometrial stromal cells treated with agents known to activate or inhibit adenylyl cyclase, protein kinase C (PKC) or phosphodiesterase (PDE). PGE(2) increased the intracellular cAMP concentration within 10 min, and this effect was attenuated by arachidonic acid and the PKC activator, 4beta-phorbol myristate acetate (PMA). The inhibitory effect of arachidonic acid on PGE(2)-induced cAMP accumulation was prevented by the PKC inhibitor, RO318425, and was absent in cells in which PKC had been downregulated by exposure to PMA for 24 h. The effect of arachidonic acid was also prevented by the PDE inhibitor, 3-isobutyl-1-methylxanthine. Arachidonic acid was shown by immunoblotting to prevent induction of cyclooxygenase-2 by PGE(2), forskolin or dibutyryl cAMP. The results indicate that arachidonic acid activates PDE through a mechanism involving PKC, counteracting a rise in intracellular cAMP in response to PGE(2). The data suggest that arachidonic acid antagonizes PGE(2) signalling through cAMP in the bovine endometrium, possibly acting to ensure a rapid return to oestrus in the case of failure of the maternal recognition of pregnancy.     

Conjugated linoleic acid reduces phorbol ester-induced prostaglandin F2alpha production by bovine endometrial cells

Recent interest in conjugated linoleic acid (CLA) research stems from the well-documented anticarcinogenic, antiatherogenic, antidiabetic, and antiobesity properties of CLA in animal models. The objective of this study was to examine the effects of 2 CLA isomers (cis-9,trans-11 and trans-10,cis-12) on phorbol 12,13-dibutyrate (PDBu)-induced PGF_2α production in cultured bovine endometrial (BEND) cells. Confluent BEND cells were incubated in the absence (control) or presence of 100 μM each of linoleic acid, cis-9,trans-11 CLA, or trans-10,cis-12 CLA for 24 h. After incubation, cells were rinsed and then stimulated with PDBu (100 ng/mL) for 6 h. Compared with untreated cells, PDBu stimulated PGF_2α secretion (+25-fold) within 6 h. The increases in PGF_2α secretion were paralleled by signifi-cant induction of prostaglandin endoperoxide synthase-2 (PGHS-2) mRNA (+63-fold) and protein (+1.6-fold) expression. In spite of stimulatory effects on PGHS-2 and peroxisome proliferator-activated receptor δ (PPARδ) mRNA responses, CLA greatly decreased PGF_2α production by PDBu-stimulated BEND cells. There was no evidence for PDBu or CLA modulation of PPARδ protein synthesis in cultured BEND cells. Results indicated that CLA modulation of PGF_2α production by BEND cells was not mediated through PGHS-2 or PPARδ gene repression.     

Immunoendocrine aspects of endometrial function and implantation

Effective ovarian and uterine function relies on a complex interplay between the endocrine and immune systems. It is generally accepted that in reproductive tissues, oestradiol and progesterone have pro- and anti-inflammatory activities respectively and, in this regard, the paracrine effects of the sex steroids on the ovary are similar to the endocrine effects on the uterus. Ovarian leukocyte recruitment and cytokine release are central to follicle development, ovulation and corpus luteum function. At the uterine level, the cyclical changes in sex steroids regulate the number and distribution of endometrial and decidual immune cells as well as other immune signalling and surveillance factors. The uterine mucosa is unique, in that it must tolerate sperm and the allogeneic blastocyst in a way that does not compromise uterine immune surveillance against bacteria, yeast and viruses. Crosstalk between the sex steroids and immune mediators (systemic and local) are central to these functions, and this article will review these mechanisms and their importance for successful reproductive function and pregnancy success.     

The protective effect of chlorogenic acid on bovine mammary epithelial cells and neutrophil function

Chlorogenic acid (CGA) is the ester of caffeic acid and quinic acid and plays an important role in antibacterial activity and anti-inflammatory properties. The objective of this study was to examine the effects of CGA on the growth of Staphylococcus aureus and the mRNA levels of the genes encoding the inflammatory response cytokines, κ-casein, and neutrophil function in bovine mammary epithelial cells (BMEC) exposed to S. aureus. Chlorogenic acid has important antibacterial, antioxidant, and anti-inflammatory functions; however, the effect of CGA on BMEC and neutrophils exposed to S. aureus has not been investigated previously. Our results demonstrated that 10, 20, and 30 μg/mL CGA had no cytotoxic effects on BMEC in culture, and that 20 μg/mL CGA enhanced the viability of BMEC exposed to S. aureus, whereas 30 μg/mL CGA reduced S. aureus growth after 9 h compared with controls. The rate of S. aureus invasion into BMEC was also attenuated by 30 μg/mL CGA compared with controls, whereas this treatment led to reduced abundance of IL6, IL8, and TLR2 mRNA in S. aureus-exposed BMEC. Migration of bovine polymorphonuclear leukocytes was significantly decreased in S. aureus-exposed BMEC with 10 and 20 μg/mL CGA treatment when compared with S. aureus treatment alone. In addition, incubation with 20 or 30 μg/mL CGA enhanced the phagocytic ability of polymorphonuclear leukocytes compared with the control group. Importantly, levels of κ-casein were enhanced by treatment of S. aureus-exposed BMEC with CGA. Our results suggest that the use of CGA may be a potent therapeutic tool against bovine mastitis caused by S. aureus.     

Dexamethasone influences endocrine and ovarian function in dairy cattle

Multiparous nonlactating Holstein cows were used to determine the effect of dexamethasone on ovarian follicular development and plasma hormone concentrations. Animals were randomly divided into two groups, control (C; n = 5) and treatment (T; n = 6), but managed as one group. Both groups were synchronized with two injections of PGF2alpha (25 mg i.m.) 11 d apart. One day after ovulation (d 0) the T group received a daily injection of dexamethasone (44 microg/kg of body weight; i.m.) until the first dominant follicle stopped growing or up to d 12 postovulation. The C group received vehicle injections. Blood samples were collected daily from all cows. Concentrations of LH and FSH did not differ between the C and T cows, whereas progesterone concentrations were lower in T than in C cows from d 4 onward. Treatment x day interaction influenced plasma insulin concentrations such that T cows had insulin concentrations 2.9- to 6.0-fold those of C cows between d 2 and 9. Dexamethasone decreased IGF-I and -II concentrations from d 5 onward. Concentrations of plasma leptin and the various IGF binding proteins were not affected by dexamethasone. Total number of follicles (> or = 5 mm) and plasma estradiol concentrations were less in T than in C cows on d 0, 1, and 4. The growth rate of the dominant follicles and maximum diameter of the dominant and subordinate follicles were not affected by dexamethasone. The diameter of the CL was 21 to 39% larger in T than in C cows between d 6 and 10. Treatment x day interaction influenced plasma cholesterol concentrations such that cholesterol levels decreased 46.8% in T cows and 19.5% in C cows between d 0 and 10. Plasma glucose concentrations were greater in T than in C cows between d 1 and 10. In summary, dexamethasone had significant effects on metabolism without a major impact on growth of the first-wave dominant follicle. Dexamethasone-induced suppression of luteal function was associated with decreased plasma IGF-I and -II concentrations.     

Differential effects of n-3 and n-6 fatty acids on prostaglandin F2alpha production by bovine endometrial cells

Recent studies have implicated n-3 polyunsaturated fatty acids in the reduction of eicosanoid production in the bovine uterus. The objective of this study was to determine whether the effect of eicosapentaenoic acid (EPA; C_20:5, n-3) on PGF_2α production by bovine endometrial (BEND) cells is influenced by the quantity of linoleic acid (C_18:2, n-6) in the incubation medium. Confluent BEND cells were incubated in the absence (control) or presence of 100 μMof EPA for 24 h. After incubation, cells were rinsed and then stimulated with phorbol 12,13-dibutyrate (PDBu; 100 ng/mL) for 6 h. Additional sets of culture dishes were treated with a combination of EPA and increasing n-6/n-3 fatty acid ratios for 24 h and then challenged with PDBu for 6 h. The PDBu stimulated PGF_2α secretion and upregulated steady-state concentrations of prostaglandin endoperoxide synthase-2 and peroxisome proliferator-activated receptor delta mRNA within 6 h. Preincubation of BEND cells with EPA for 24 h decreased PGF_2α response to phorbol ester, but had no detectable effects on prostaglandin endoperoxide synthase-2 or peroxisome proliferator-activated receptor delta mRNA abundance in PDBu-stimulated BEND cells. The inhibitory effect of EPA on PGF_2α production was reverted in BEND cells treated with an increasing n-6-to-n-3 fatty acid ratio. Findings indicate that the net inhibition of endometrial PGF_2α bioynthesis by n-3 fatty acids may vary depending on the ratio of n-6 to n-3 fatty acids in the uterus.     

Effect of calf housing on plasma ascorbate and endocrine and immune function

The effect of housing calves in indoor metal pens (1.2 m x 1.2 m) or commercial calf hutches was determined on plasma concentration of cortisol, antibodies, and ascorbate. Six calves per treatment were deprived of colostrum, assigned randomly to treatment, and fed a commercial milk replacer until 56 d of age. Calves housed in hutches had higher plasma IgG concentrations than calves in pens at 42 and 56 d of age. Housing had no effect on plasma IgM concentration. Antibody titers to keyhole limpet hemocyanin injected at 14 and 28 d of age were higher in hutch-housed calves from 21 to 56 d of age. Calves housed in hutches also had lower plasma cortisol concentrations, although cortisol decreased with age in both treatment groups. Plasma ascorbate and ascorbate plus dehydroascorbate were higher in hutch-housed calves. Regression analysis indicated a positive relationship between plasma ascorbate and IgG in metal penhoused calves and a negative relationship in hutch-housed calves. Housing in 1.2 m x 1.2 m metal pens decreases cortisol, plasma ascorbate, IgG, and specific antibody titers in dairy calves relative to calves housed in hutches.     

Host-pathogen interactions in bovine mammary epithelial cells and HeLa cells by Staphylococcus aureus isolated from subclinical bovine mastitis

Staphylococcus aureus is a common pathogen that causes subclinical bovine mastitis due to several virulence factors. In this study, we analyzed S. aureus isolates collected from the milk of cows with subclinical mastitis that had 8 possible combinations of bap, icaA, and icaD genes, to determine their capacity to produce biofilm on biotic (bovine primary mammary epithelial cells and HeLa cells) and abiotic (polystyrene microplates) surfaces, and their ability to adhere to and invade these cells. We also characterized isolates for microbial surface components recognizing adhesive matrix molecules (MSCRAMM) and agr genes, and for their susceptibility to cefquinome sulfate in the presence of biofilm. All isolates adhered to and invaded both cell types, but invasion indexes were higher in bovine primary mammary epithelial cells. Using tryptic soy broth + 1% glucose on abiotic surfaces, 5 out of 8 isolates were biofilm producers, but only the bap⁺icaA⁺icaD⁺ isolate was positive in Dulbecco's Modified Eagle's medium. The production of biofilm on biotic surfaces occurred only with this isolate and only on HeLa cells, because the invasion index for bovine primary mammary epithelial cells was too high, making it impossible to use these cells in this assay. Of the 5 biofilm producers in tryptic soy broth + 1% glucose, 4 presented with the bap/fnbA/clfA/clfB/eno/fib/ebpS combination, and all were protected from cefquinome sulfate. We found no predominance of any agr group. The high invasive potential of S. aureus made it impossible to observe biofilm in bovine primary mammary epithelial cells, and we concluded that cells with lower invasion rates, such as HeLa cells, were more appropriate for this assay.     

Effects of induced clinical mastitis during preovulation on endocrine and follicular function

The objective of the study was to determine if experimentally induced clinical mastitis before ovulation resulted in alterations of endocrine function, follicular growth, or ovulation. On d 8 (estrus = d 0), cows were challenged (TRT; n = 19) with Streptococcus uberis or were not challenged (control; n = 14). Forty-eight hours after induction of luteal regression on d 12, blood samples were collected to determine estradiol-17β, LH pulse frequency, and occurrence of the LH surge. Ovaries were scanned to monitor follicular growth and ovulation. Cows with clinical mastitis (n = 12) had elevated rectal temperatures, somatic cell counts, and mammary scores. Estrus and ovulation occurred in 4 of 12 clinically infected cows and in all control cows. Cows that were challenged but did not develop clinical mastitis (n = 5) displayed estrus and ovulated. Due to differences in expression of estrus, cows were further subdivided for analyses into 4 groups: control, TRT-EST (infected cows that displayed estrus; n = 4), TRT-NOEST (infected cows that did not display estrus; n = 8), and NOMAS (cows that were inoculated but did not develop mastitis; n = 4). Ovulation rate was 100% for CON, NOMAS, and TRT-EST compared with 0% for TRT-NOEST cows. Size of the ovulatory follicle (“presumed” ovulatory follicle in TRT-NOEST cows) was similar for all groups. Frequency of LH pulses was decreased in TRT-NOEST compared with CON, TRT-EST, and NO-MAS. Estradiol-17β increased over time in CON, NO-MAS, and TRT-EST cows, but did not increase in TRT-NOEST cows. Cows with clinical mastitis may exhibit estrus and ovulate normally or have disruptions in normal physiology including decreased LH pulsatility, absence of an LH surge and estrous behavior, suppressed estradiol-17β, and failure to ovulate.     

S+4和S-4同分异构体的密度泛函研究

用分子图形软件分别设计出S4 ,S4-离子的20种同分异构体构型,使用B3P86密度泛函进行几何构型优化和振动频率计算,分别得到了它们的分子结构特点:电子态、对称性、谐振频率和总能量.分子大多以二配位成键,也有采用一、三配位成键的,但总能量较高,根据分子的总能量得出最稳定的同分异构体.     

Effect of endocrine and paracrine factors on protein synthesis and cell proliferation in bovine hoof tissue culture

Laminitis is a major cause of lameness in dairy cattle, and is widely attributed to a defect in the horny tissue that gives the hoof its mechanical strength. Defective horn is associated with, and may be preceded by, impaired keratin deposition in the hoof epidermis. The cause of abnormal keratin deposition is not easily identified but, like epidermal keratinization in other tissues, is likely to be controlled by hormones and the paracrine action of locally produced growth factors. The hormonal regulation of keratin synthesis and cell proliferation in the bovine hoof was studied using tissue explants in organ culture. As the highest incidence of laminitis is in early lactation, the study focused on insulin, cortisol and prolactin, three hormones implicated in lactogenesis and galactopoiesis. Incubation of tissue explants for 24 h in medium containing insulin (10-5000 ng/ml) stimulated protein synthesis measured by incorporation of 35S-labelled amino acids. Histochemical examination showed that insulin binding co-localized with the site of protein synthesis. Insulin also stimulated DNA synthesis, an index of cell proliferation, which was measured by incorporation of [3H]methyl thymidine. Cortisol (10-5000 ng/ml) decreased protein synthesis, whereas prolactin (10-5000 ng/ml) had no significant effect on protein or DNA synthesis. Epidermal growth factor (10-200 ng/ml), a potent inhibitor of keratinization in other tissues, stimulated protein synthesis compared with untreated controls. Epidermal growth factor binding was located microscopically to the germinal and differentiating epidermal layers. SDS-PAGE and fluorography showed that the population of proteins synthesized in the presence of any hormone or growth factor combination did not differ from that in untreated controls and included the keratins involved in horn deposition. The results show that bovine hoof keratinization is under endocrine and growth factor control, and suggest that systemic changes in lactogenic hormones may act to inhibit keratin deposition.