Bovine follicle development is associated with divergent changes in activin-A, inhibin-A and follistatin and the relative abundance of different follistatin isoforms in follicular fluid

The aim was to determine whether follicle growth in cattle is accompanied by changes in levels of inhibin-A (inh-A), activin-A (act-A) and different Mr isofomus of follistatin (FS) in bovine follicular fluid (bFF), reflecting differential roles of these proteins during folliculogenesis. Follcles (n= 146) from 2-20 min diameter were dissected from ovaries of similar to 40 cattle. Immunoassays were used to measure total FS, act-A, inh-A, oestradiol (E) and progesterone (P) levels; immunoblotting was used to quanti, the relative abundance of different FS isoforms. Follicle growth from 2-6 mm was associated with a 6-fold increase in inh-A and 30-fold increase in act-A; FS remained uniformly high from 2-10 turn. From 6-2 min, inh-A remained high while act-A and FS fell 3-fold and 2-fold, respectively. Act-A/FS ratio increased 20-fold from 2-6 mm before falling slightly through to 20 mm. Act-A/inh-A ratio increased 6-fold from 2-6 nun before falling 2-fold from 6 to 17-20 mm. These findings imply a marked increase in relative activin 'tone' around the stage at which dominant follicle,;election occurs. When larger follicles (13-20 mm) were subdivided according to E/P ratio, those with high (> 5) E/P ratio had lower (2-fold; P < 0(.)001) levels of inh-A and act-A in comparison to follicles with low (< 5) E/P ratio, but there were no significant diffierences in FS, act-A/inh-A ratio or act-A/FS ratio. Thus follicle size, but not oestrogenic status, has a major influence on the intrafollicular balance between act-A and its opposing factors, inh-A and FS. Six FS isoforms were detected in bFF (apparent Mr: 65, 41, 37, 35, 33 and 31 kDa) averaging 6, 13, 24, 26 13 and 17% respectively of total FS. During growth from 2-20 mm the proportion of total FS represented by 605, 41 and 37 kDa isoforms increased similar to 2-fold while the proportion represented by the 33 and 31 kDa isoforms decreased by 3-fold and 1(.)6-fold, respectively. Treatment of bovine granulosa cells in vitro with FSH and IGF alone or in combination increased total FS secretion up to 12-fold but did not affect the relative abundance of the five different FS isoforms detected. While the functional significance of the intriguing shift in FS isoform abundance in bFF during follicle development remains to be established, we have shown that a marked increase in intrafollicular activin 'tone' accompanies bovine follicle growth from 3-6 min, corresponding to the stage at which the FSH-dependent follicle selection mechanism operates in this species.

Seasonal variations in the developmental competence of bovine oocytes matured in vitro

Ovaries were collected over a period of two years from heifers slaughtered at under 30 months of age and used to harvest 1757 oocytes. After in vitro maturation, fertilisation and culture, the proportions of oocytes and cleaved embryos that developed to blastocysts were significantly higher (P < 0.01) in the autumn, from September to November, than in the spring, from March to May. In contrast, embryo development, as assessed by oocytes that developed to eight or more cells and blastocysts, was lowest (P < 0.01) in the spring. These results were consistent during the two-year study, indicating a seasonal fluctuation in oocyte competence.

Ovarian follicle development in the laying hen is accompanied by divergent changes in inhibin A, inhibin B, activin A and follistatin production in granulosa and theca layers

To study the potential involvement of inhibin A (inhA), inhibin B (inhB), activin A (actA) and follistatin (FS) in the recruitment of follicles into the preovulatory hierarchy, growing follicles (ranging from 1 mm to the largest designated F1) and the three most recent postovulatory follicles (POFs) were recovered from laying hens (n=11). With the exception of <4 mm follicles and POFs, follicle walls were dissected into separate granulosa (G) and theca (T) layers before extraction. Contents of inhA, inhB, actA and FS in tissue extracts were assayed using specific two-site ELISAs and results are expressed per mg DNA. InhB content of both G and T followed a similar developmental pattern, although the content was >4-fold higher in G than in T at all stages. InhB content was very low in follicles <4 nun but increased - 50-fold (P<0.0001) to peak in 7-9 mm follicles, before falling steadily as follicles entered and moved up the follicular hierarchy (40-fold; 8 mm vs F2). In stark contrast, inhA remained very low in prehierarchical follicles (&LE; 9 mm) but then increased progressively as follicles moved up the preovulatory hierarchy to peak in F1 (&SIM; 100-fold increase; P<0.0001); In F1 >97% of inhA was confined to the G layer whereas in 5-9 mm follicles inhA was only. detected in the T layer. Both inhA and inhB contents of POFs were significantly reduced compared with F1. Follicular actA was mainly confined to the T layer although detectable levels were present in G from 9 nun; actA was low between 1 and 9 mm but increased sharply as follicles entered the preovulatory hierarchy (&SIM;6-fold higher in F4; P<0.0001); levels then fell &SIM;2-fold as the follicle progressed to F1. Like actA, FS predominated in the T although significant amounts were also present in the G of prehierarchical follicles (4-9 mm), in contrast to actA, which was absent from the G. The FS content of T rose &SIM;3-fold from 6 mm to a plateau which was sustained until F1. In contrast, the FS content of G was greatest in prehierarchical follicles and fell &SIM;4-fold in F4-F1 follicles. ActA and FS contents of POFs were reduced compared with F1. In vitro studies on follicle wall explants confirmed the striking divergence in the secretion of inhA and inhB during follicle development. These findings of marked stage-dependent differences in the expression of inhA, inhB, actA and FS proteins imply a significant functional role for these peptides in the recruitment and ordered progression of follicles within the avian ovary.

Probiotics and cancer: from in vitro to human studies

Studies in cell cultures and animal models provide evidence that probiotics can beneficially influence various stages in development of colon cancer including tumor initiation, promotion and metastasis. For example, oral administration of Lactobacillus and Bifidobacterium strains can prevent genotoxic damage to the colonic epithelium (considered to be an early stage of the carcinogenic process). Administration to rats of probiotics reduced the incidence of carcinogen-induced pre-cancerous lesions (aberrant crypt foci) in the colon. Furthermore a combination of Bifidobacterium longum and inulin (a prebiotic) was more effective than either treatment alone. In this latter study, the dietary treatments were given after exposure to the carcinogen, which suggests that the protective effects were being exerted at the promotional phase of carcinogenesis. L. acidophilus feeding has been shown to decrease the incidence of colon tumors in rats challenged with a carcinogen and B. longum reduced the incidence of carcinogeninduced colon, liver and mammary tumors. There is limited evidence from epidemiological studies for protective effects of products containing probiotics in humans, but a number of recent dietary intervention studies in healthy subjects and in polyp and cancer patients have yielded promising results on the basis of biomarkers of cancer risk and grade of colorectal tumors.

Development of a fermentation system to model sessile bacterial populations in the human colon

A fermentation system was designed to model the human colonic microflora in vitro. The system provided a framework of mucin beads to encourage the adhesion of bacteria, which was encased within a dialysis membrane. The void between the beads was inoculated with faeces from human donors. Water and metabolites were removed from the fermentation by osmosis using a solution of polyethylene glycol (PEG). The system was concomitantly inoculated alongside a conventional single-stage chemostat. Three fermentations were carried out using inocula from three healthy human donors. Bacterial populations from the chemostat and biofilm system were enumerated using fluorescence in situ hybridization. The culture fluid was also analysed for its short-chain fatty acid (SCFA) content. A higher cell density was achieved in the biofilm fermentation system (taking into account the contribution made by the bead-associated bacteria) as compared with the chemostat, owing to the removal of water and metabolites. Evaluation of the bacterial populations revealed that the biofilm system was able to support two distinct groups of bacteria: bacteria growing in association with the mucin beads and planktonic bacteria in the culture fluid. Furthermore, distinct differences were observed between populations in the biofilm fermenter system and the chemostat, with the former supporting higher populations of clostridia and Escherichia coli. SCFA levels were lower in the biofilm system than in the chemostat, as in the former they were removed via the osmotic effect of the PEG. These experiments demonstrated the potential usefulness of the biofilm system for investigating the complexity of the human colonic microflora and the contribution made by sessile bacterial populations.

Development of a quantitative tool for the comparison of the prebiotic effect of dietary oligosaccharides

Aims: To develop a quantitative equation [prebiotic index ( PI)] to aid the analysis of prebiotic fermentation of commercially available and novel prebiotic carbohydrates in vitro, using previously published fermentation data. Methods: The PI equation is based on the changes in key bacterial groups during fermentation. The bacterial groups incorporated into this PI equation were bifidobacteria, lactobacilli, clostridia and bacteroides. The changes in these bacterial groups from previous studies were entered into the PI equation in order to determine a quantitative PI score. PI scores were than compared with the qualitative conclusions made in these publications. In general the PI scores agreed with the qualitative conclusions drawn and provided a quantitative measure. Conclusions: The PI allows the magnitude of prebiotic effects to be quantified rather than evaluations being solely qualitative. Significance and Impact of the Study: The PI equation may be of great use in quantifying prebiotic effects in vitro. It is expected that this will facilitate more rational food product development and the development of more potent prebiotics with activity at lower doses.

Anti-cancer properties of phenolics from apple waste on colon carcinogenesis in vitro

Colorectal cancer is one of the most common cancers in Western countries. The World Health Organisation identifies diet as a critical risk factor in the development and progression of this disease and the protective role of high levels of fruit and vegetable consumption. Several studies have shown that apples contain several phenolic compounds that are potent anti-oxidants in humans. However, little is known about other beneficial properties of apple phenolics in cancer. We have used the HT29, HT115 and CaCo-2 cell lines as in vitro models to examine the effect of apple phenolics (0.01–0.1% apple extract) on key stages of colorectal carcinogenesis, namely; DNA damage (Comet assay), colonic barrier function (TER assay), cell cycle progression (DNA content assay) and invasion (Matrigel assay). Our results indicate that a crude extract of apple phenolics can protect against DNA damage, improve barrier function and inhibit invasion (p < 0.05). The anti-invasive effects of the extract were enhanced with twenty-four hour pretreatment of cells (p < 0.05). We have shown that a crude apple extract from waste, rich in phenolic compounds, beneficially influences key stages of carcinogenesis in colon cells in vitro.

Enterolactone restricts the proliferation of the LNCaP human prostate cancer cell line in vitro

Ecological data suggest a long-term diet high in plant material rich in biologically active compounds, such as the lignans, can significantly influence the development of prostate cancer over the lifetime of an individual. The capacity of a pure mammalian lignan, enterolactone (ENL), to influence the proliferation of the LNCaP human prostate cancer cell line was investigated as a function of cell density, metabolic activity, expression and secretion of prostate specific antigen (PSA), cell cycle profile, and the expression of genes involved in development and progression of prostate cancer. Treatment with a subcytotoxic concentration of ENL (60 mu M for 72 h) was found to reduce: cell density (57.5%, SD 7.23, p < 0.001), metabolic activity (55%, SD 0.03, p < 0.001), secretion of PSA (48.50% SD 4.74, p = 0.05) and induce apoptosis (8.33-fold SD 0.04, p = 0.001) compared to untreated cells. Cotreatment with 10 mu M etoposide was found to increase apoptosis by 50.17% (SD 0.02, p < 0.001). Additionally, several key genes (e.g. MCMs, survivin and CDKs) were beneficially regulated by ENL treatment (p < 0.05). The data suggest that the antiproliferative activity of ENL is a consequence of altered expression of cell cycle associated genes and provides novel molecular evidence for the antiproliferative properties of a pure lignan in prostate cancer.

Development of multi-electrode array screening for anticonvulsants in acute rat brain slices

The acute hippocampal brain slice preparation is an important in vitro screening tool for potential anticonvulsants. Application of 4-aminopyridine (4-AP) or removal of external Mg2+ ions induces epileptiform bursting in slices which is analogous to electrical brain activity seen in status epilepticus states. We have developed these epileptiform models for use with multi-electrode arrays (MEAs), allowing recording across the hippocampal slice surface from 59 points. We present validation of this novel approach and analyses using two anticonvulsants, felbamate and phenobarbital, the effects of which have already been assessed in these models using conventional extracellular recordings. In addition to assessing drug effects on commonly described parameters (duration, amplitude and frequency), we describe novel methods using the MEA to assess burst propagation speeds and the underlying frequencies that contribute to the epileptiform activity seen. Contour plots are also used as a method of illustrating burst activity. Finally, we describe hitherto unreported properties of epileptiform, bursting induced by 100 mu M 4-AP or removal of external Mg2+ ions. Specifically, we observed decreases over time in burst amplitude and increase over time in burst frequency in the absence of additional pharmacological interventions. These MEA methods enhance the depth, quality and range of data that can be derived from the hippocampal slice preparation compared to conventional extracellular recordings. it may also uncover additional modes of action that contribute to anti-epileptiform drug effects. (C) 2009 Elsevier B.V. All rights reserved.

Changes in expression of bone morphogenetic proteins (BMPs), their receptors and inhibin co-receptor betaglycan during bovine antral follicle development: inhibin can antagonize the suppressive effect of BMPs on thecal androgen production

We reported previously that bone morphogenetic proteins (BMPs) potently suppress CYP17 expression and androgen production by bovine theca interna cells (TC) in vitro. In this study, real-time PCR was used to analyse gene expression in TC and granulosa cell (GC) layers from developing bovine antral follicles (1-18 mm). Abundance of mRNA transcripts for four BMPs (BMP2, BMP4, BMP6, and BMP7) and associated type I (BMPR1A, BMPR1B, ACVR1 and ACVR1B) and type II (BMPR2, ACVR2A and ACVR2B) receptors showed relatively modest, though significant, changes during follicle development. BMP2 was selectively expressed in GC, while BMP6, BMP7 and betaglycan (TGFBR3) were more abundant in TC. Abundance of betaglycan mRNA (inhibin co-receptor) in TC increased progressively (fivefold; P<0.001) as follicles grew from 1-2 to 9-10 mm. This suggests a shift in thecal responsiveness to GC-derived inhibin, produced in increasing amounts as follicles achieve dominance. This prompted us to investigate whether inhibin can function as a physiological antagonist of BMP action on bovine TC in vitro, in a manner comparable to that for activin signalling. BMP4, BMP6 and BMP7 abolished LH-induced androstenedione secretion and suppressed CYP17 mRNA >200-fold (P<0.001), while co-treatment with inhibin-A reversed the suppressive action of BMP in each case (P<0.001). Results support a physiological role for granulosa-derived inhibin as an antagonist of BMP action on thecal androgen synthesis. A shift in intrafollicular balance between thecal BMP signalling (inhibitory for androgen synthesis) and betaglycan-dependent inhibin signalling (stimulatory for androgen synthesis) accords with the physiological requirement to deliver an adequate supply of aromatase substrate to GC of developing follicles.

Response of porcine intestinal in vitro organ culture tissues following exposure to Lactobacillus plantarum JC1 and Salmonella Typhimurium SL1344.

The development of novel intervention strategies for the control of zoonoses caused by bacteria such as Salmonella spp. in livestock requires appropriate experimental models to assess their suitability. Here, a novel porcine intestinal in vitro organ culture (IVOC) model utilizing cell crown (CC) technology (CCIVOC) (Scaffdex) was developed. The CCIVOC model was employed to investigate the characteristics of association of S. enterica serovar Typhimurium strain SL1344 with porcine intestinal tissue following exposure to a Lactobacillus plantarum strain. The association of bacteria to host cells was examined by light microscopy and electron microscopy (EM) after appropriate treatments and staining, while changes in the proteome of porcine jejunal tissues were investigated using quantitative label-free proteomics. Exposure of porcine intestinal mucosal tissues to L. plantarum JC1 did not reduce the numbers of S. Typhimurium bacteria associating to the tissues but was associated with significant (P < 0.005) reductions in the percentages of areas of intestinal IVOC tissues giving positive staining results for acidic mucins. Conversely, the quantity of neutrally charged mucins present within the goblet cells of the IVOC tissues increased significantly (P < 0.05). In addition, tubulin- was expressed at high levels following inoculation of jejunal IVOC tissues with L. plantarum. Although L. plantarum JC1 did not reduce the association of S. Typhimurium strain SL1344 to the jejunal IVOC tissues, detection of increased acidic mucin secretion, host cytoskeletal rearrangements, and proteins involved in the porcine immune response demonstrated that this strain of L. plantarum may contribute to protecting the pig from infections by S. Typhimurium or other pathogens.

In vitro fermentation of potential prebiotic flours from natural sources: impact on the human colonic microbiota and metabolome

Scope: Fibers and prebiotics represent a useful dietary approach for modulating the human gut microbiome. Therefore, aim of the present study was to investigate the impact of four flours (wholegrain rye, wholegrain wheat, chickpeas and lentils 50:50, and barley milled grains), characterized by a naturally high content in dietary fibers, on the intestinal microbiota composition and metabolomic output. Methods and results: A validated three-stage continuous fermentative system simulating the human colon was used to resemble the complexity and diversity of the intestinal microbiota. Fluorescence in situ hybridization was used to evaluate the impact of the flours on the composition of the microbiota, while small-molecule metabolome was assessed by NMR analysis followed by multivariate pattern recognition techniques. HT29 cell-growth curve assay was used to evaluate the modulatory properties of the bacterial metabolites on the growth of intestinal epithelial cells. All the four flours showed positive modulations of the microbiota composition and metabolic activity. Furthermore, none of the flours influenced the growth-modulatory potential of the metabolites toward HT29 cells. Conclusion: Our findings support the utilization of the tested ingredients in the development of a variety of potentially prebiotic food products aimed at improving gastrointestinal health.

Ovarian expression of insulin-like peptide 3 (INSL3) and its receptor (RXFP2) during development of bovine antral follicles and corpora lutea and measurement of circulating INSL3 levels during synchronized estrous cycles

Insulin-like peptide 3 (INSL3), a major product of testicular Leydig cells, is also expressed by the ovary but its functional role remains poorly understood. Here, we quantified expression of INSL3 and its receptor RXFP2 in theca interna (TIC) and granulosa (GC) compartments of developing bovine antral follicles and in corpora lutea (CL). INSL3 and RXFP2 mRNA levels were much higher in TIC than GC and increased progressively during follicle maturation with INSL3 peaking in large (11-18mm) estrogen-active follicles and RXFP2 peaking in 9-10mm follicles before declining in larger (11-18mm) follicles. Expression of both INSL3 and RXFP2 in CL was much lower than in TIC. In situ hybridization and immunohistochemistry confirmed abundant expression of INSL3 mRNA and protein in TIC. These observations indicate follicular TIC rather than CL as the primary site of both INSL3 production and action, implying a predominantly auto-/paracrine role in TIC. To corroborate the above findings, we showed that in vitro exposure of TIC to a luteinizing concentration of LH greatly attenuated expression of both INSL3 and its receptor while increasing progesterone secretion and expression of STAR and CYP11A1. Moreover, in vivo, a significant cyclic variation in plasma INSL3 was observed during synchronized estrous cycles. INSL3 and estradiol-17β followed a similar pattern, both increasing after luteolysis, before falling sharply after the LH surge. Thus, theca-derived INSL3, likely from the dominant pre-ovulatory follicle, is detectable in peripheral blood of cattle and expression is down-regulated during luteinisation induced by the pre-ovulatory LH surge. Collectively, these findings underscore the likely role of INSL3 as an important intrafollicular modulator of TIC function/steroidogenesis, whilst raising doubts about its potential contribution to CL function.

Development of antimicrobial synbiotics using potentially-probiotic faecal isolates of Lactobacillus fermentum and Bifidobacterium longum

The aims of the present study were to investigate in vitro the antimicrobial activity of Lactobacillus fermentum and Bifidobacterium longum, isolated from faeces of healthy elderly individuals, against enterohaemorrhagic Escherichia coli (E. coli O157:H7) and enteropathogenic E. coli (E. coli O86), to determine the capability of the selected strains to tolerate acid and bile in vitro, to select suitable carbohydrates in order to enhance the growth and maximise antimicrobial activity of the putative probiotic organisms and examine the adhesion properties of the synbiotics. Antimicrobial activity of the putative probiotics and synbiotics was investigated by a microtitre method using cell-free culture supernatants (CFCS). Results of the antimicrobial assay showed that both putative probiotic strains produced compounds at pH 5 that lead to higher lag phases of both E. coli O157:H7 and E. coli O86. When half the quantity of cell-free culture supernatants of both probiotic strains was used at pH 5, B. longum maintained the same antimicrobial effect against both strains of E. coli, whereas L. fermentum lead to a higher lag phase of E. coli O86 only. Neutralization of the culture supernatants with alkali reduced the antimicrobial effect with only cell-free supernatant of L. fermentum causing lower maximum growth rates of E. coli O157:H7 and E. coli O86. L. fermentum appeared to be acid tolerant whereas B. longum was more susceptible to acid and both isolates were bile tolerant. A short chain fructooligosaccharide (scFOS) and an isomalto-oligosaccharide (IMO) proved to be the most effective substrates, enhancing antimicrobial activity for L. fermentum and B. longum respectively. The adhesion of the synbiotic combinations showed that L. fermentum, exhibited higher percentage of adhesion when grown on glucose and as a synbiotic combination with scFOS whereas B. longum exhibited lowest percentage of adhesion when grown on both glucose and IMO.

Overexpression of coconut AINTEGUMENTA-like gene, CnANT, promotes in vitro regeneration in transgenic Arabidopsis

Knowledge of the molecular biological changes underlying the process of embryogenesis is important for the improvement of somatic embryogenesis of coconut. Among the transcription factors that control the transition from vegetative to embryogenic growth, members of APETALA2/Ethylene-responsive element binding protein domain family play an important role in promoting embryo development. Significant insights into the role of AP2 genes have been obtained by the ectopic expression of AP2 sub family genes in transgenic Arabidopsis. A homolog of the AINTEGUMENTA-like gene that encodes the two AP2 domains and the linker region was identified in the coconut genome. Phylogenetic analysis showed that this gene, CnANT, encodes a protein that branched with BABY BOOM/PLETHORA clade in the AINTEGUMENTA-like major clade and was similar to the oil palm EgAP2-1 protein. According to real time RT-PCR results, higher expression of CnANT was observed in more mature zygotic embryos. Also, high CnANT expression was recorded in embryogenic callus compared to other stages of somatic embryogenesis. We examined the effect of ectopic CnANT expression on the development and regenerative capacity of transgenic Arabidopsis. Overexpression of CnANT in Arabidopsis induced hormone free regeneration of explants. Furthermore, ectopic expression of CnANT enhanced regeneration in vitro and suggested a role for this gene in cell proliferation during in vitro culture.