Nathan J. Bivens

Transcriptional Profiling of Pig Embryogenesis by Using a 15-K Member Unigene Set Specific for Pig Reproductive Tissues and Embryos

Abstract Differential mRNA expression patterns were evaluated between germinal vesicle oocytes (pgvo), four-cell (p4civv), blastocyst (pblivv), and in vitro-produced four-cell (p4civp) and in vitro-produced blastocyst (pblivp) stage embryos to determine key transcripts responsible for early embryonic development in the pig. Five comparisons were made: pgvo to p4civv, p4civv to pblivv, pgvo to pblivv, p4civv to p4civp, and pblivv to pblivp. ANOVA (P < 0.05) was performed with the Benjamini and Hochberg false-discovery-rate multiple correction test on each comparison. A comparison of pgvo to p4civv, p4civv to pblivv, and pgvo to pblivv resulted in 3214, 1989, and 4528 differentially detected cDNAs, respectively. Real-time PCR analysis on seven transcripts showed an identical pattern of changes in expression as observed on the microarrays, while one transcript deviated at a single cell stage. There were 1409 and 1696 differentially detected cDNAs between the in vitro- and in vivo-produced embryos at the four-cell and blastocyst stages, respectively, without the Benjamini and Hochberg false-discovery-rate multiple correction test. Real-time polymerase chain reaction (PCR) analysis on four genes at the four-cell stage showed an identical pattern of gene expression as found on the microarrays. Real-time PCR analysis on four of five genes at the blastocyst stage showed an identical pattern of gene expression as found on the microarrays. Thus, only 1 of the 39 comparisons of the pattern of gene expression exhibited a major deviation between the microarray and the real-time PCR. These results illustrate the complex mechanisms involved in pig early embryonic development.

Effects of Vendor and Genetic Background on the Composition of the Fecal Microbiota of Inbred Mice

The commensal gut microbiota has been implicated as a determinant in several human diseases and conditions. There is mounting evidence that the gut microbiota of laboratory mice (Mus musculus) similarly modulates the phenotype of mouse models used to study human disease and development. While differing model phenotypes have been reported using mice purchased from different vendors, the composition and uniformity of the fecal microbiota in mice of various genetic backgrounds from different vendors is unclear. Using culture-independent methods and robust statistical analysis, we demonstrate significant differences in the richness and diversity of fecal microbial populations in mice purchased from two large commercial vendors. Moreover, the abundance of many operational taxonomic units, often identified to the species level, as well as several higher taxa, differed in vendor- and strain-dependent manners. Such differences were evident in the fecal microbiota of weanling mice and persisted throughout the study, to twenty-four weeks of age. These data provide the first in-depth analysis of the developmental trajectory of the fecal microbiota in mice from different vendors, and a starting point from which researchers may be able to refine animal models affected by differences in the gut microbiota and thus possibly reduce the number of animals required to perform studies with sufficient statistical power.

Stimulation of radial expansion in arabidopsis roots by inhibitors of actomyosin and vesicle secretion but not by various inhibitors of metabolism

Plant morphogenesis depends on accurate control over growth anisotropy. To learn to what extent the control of growth anisotropy depends on cellular metaolism, we surveyed the response of growing roots to a range of inhibitors. Seedlings of Arabidopsis thaliana L. (Heynh), 7–8 d old, were transplanted onto plates containing an inhibitor, and elongation and radial expansion of roots were measured over the subsequent 2-d period. Fourteen inhibitors of diverse metabolic processes inhibited root elongation but failed to stimulate radial expansion. These inhibitors were aluminum sulfate, aphidicolin (DNA synthesis), caffeine (cell-plate formation), cisplatin (DNA synthesis), cycloheximide (protein synthesis), 3,4-dehydro-l-proline (proline hydroxylation), 6-dimethylaminopurine (protein kinases), dinitrophenol (mitochondrial ATP synthesis), galactose (UDP-glucose formation), Lovastatin, formerly mevinolin (isoprenoid formation), methionine sulfoximine (glutamine synthetase), methotrexate (folate metabolism), XRD-489 (synthesis of branched-chain amino acids), and high or low calcium treatments. These results show that various types of metabolic disruption, although inhibitory to elongation, do not reduce the high degree of anisotropic growth of the root. However, five chemicals did stimulate radial expansion; namely, the detergent, digitonin; two inhibitors of vesicle secretion, monensin and brefeldin A; and two inhibitors of actomyosin, cytochalasin B and butanedione monoxime. The maximum radial expansion induced by these compounds (except butanedione monoxime) was greater than that caused by ethylene, and the morphology of treated roots did not resemble that of roots treated with ethylene. These results indicate that vesicle secretion and actomyosin play a role in controlling anisotropic expansion.

Developmental Expression of 2489 Gene Clusters During Pig Embryogenesis: An Expressed Sequence Tag Project

Abstract Identification of mRNAs that are present at early stages of embryogenesis is critical for a better understanding of development. To this end, cDNA libraries were constructed from germinal vesicle-stage oocytes, in vivo-produced four-cell- and blastocyst-stage embryos, and from in vitro-produced four-cell- and blastocyst-stage embryos. Randomly picked clones (10 848) were sequenced from the 3′ end and those of sufficient quality (8066, 74%) were clustered into groups of sequence similarity (>95% identity), resulting in 2489 clusters. The sequence of the longest representative expressed sequence tag (EST) of each cluster was compared with GenBank and TIGR. Scores below 200 were considered unique, and 1114 (44.8%) did not have a match in either database. Sequencing from the 5′ end yielded 12 of 37 useful annotations, suggesting that one third of the 1114 might be identifiable, still leaving over 700 unique ESTs. Virtual Northerns compared between the stages identified numerous genes where expression appears to change from the germinal vesicle oocyte to the four-cell stage, from the four-cell to blastocyst stage, and between in vitro- and in vivo-derived four-cell- and blastocyst-stage embryos. This is the first large-scale sequencing project on early pig embryogenesis and has resulted in the discovery of a large number of genes as well as possible stage-specific expression. Because many of these ESTs appear to not be in the public databases, their addition will be useful for transcriptional profiling experiments conducted on early pig embryos.

Effects of exposure to bisphenol A and ethinyl estradiol on the gut microbiota of parents and their offspring in a rodent model

ABSTRACT Gut dysbiosis may result in various diseases, such as metabolic and neurobehavioral disorders. Exposure to endocrine disrupting chemicals (EDCs), including bisphenol A (BPA) and ethinyl estradiol (EE), especially during development, may also increase the risk for such disorders. An unexplored possibility is that EDC-exposure might alter the gut microbial composition. Gut flora and their products may thus be mediating factors for the disease-causing effects of these chemicals. To examine the effects of EDCs on the gut microbiome, female and male monogamous and biparental California mice (Peromyscus californicus) were exposed to BPA (50 mg/kg feed weight) or EE (0.1 ppb) or control diet from periconception through weaning. 16s rRNA sequencing was performed on bacterial DNA isolated from fecal samples, and analyses performed for P0 and F1 males and females. Both BPA and EE induced generational and sex-dependent gut microbiome changes. Many of the bacteria, e.g. Bacteroides, Mollicutes, Prevotellaceae, Erysipelotrichaceae, Akkermansia, Methanobrevibacter, Sutterella, whose proportions increase with exposure to BPA or EE in the P0 or F1 generation are associated with different disorders, such as inflammatory bowel disease (IBD), metabolic disorders, and colorectal cancer. However, the proportion of the beneficial bacterium, Bifidobacterium, was also elevated in fecal samples of BPA- and EE-exposed F1 females. Intestinal flora alterations were also linked to changes in various metabolic and other pathways. Thus, BPA and EE exposure may disrupt the normal gut flora, which may in turn result in systemic effects. Probiotic supplementation might be an effective means to mitigate disease-promoting effects of these chemicals.

Large-scale generation and analysis of expressed sequence tags from porcine ovary.

Abstract One method to identify the factors that control ovarian function is to characterize the genes that are expressed in ovary. In the present study, cDNA libraries from fetal, neonatal, and prepubertal porcine ovaries, pubertal ovaries on different days of the estrous cycle (Days 0 [follicle], 5, and 12 [follicle and corpus luteum]), and follicles isolated from weaned sows (diameter, 2, 4, 6, and 8 mm) were constructed and sequenced. A total of 22 176 cDNAs were sequenced, of which 15 613 were of sufficient quality for clustering. Clustering of cDNAs resulted in 8507 contigs, 6294 (74%) of which were comprised of a single sequence. Sixty-eight percent of the contigs had consensus sequences that were homologous to existing Tentative Consensus (TC) sequences or mature transcripts (ET) in The Institute for Genomic Research Porcine Gene Index. The consensus sequences were classified according to the Gene Ontology Index. Most cDNA-encoded proteins were components of the nucleus, ribosome, or mitochondrion. The proteins primarily functioned in binding, catalysis, and transport. Nearly 75% of the proteins were involved in metabolism and cell growth and/or maintenance. Analysis of the cDNA frequency across different libraries demonstrated differential gene expression within different-size follicles, between follicles and corpora lutea, and across developmental time-points. The expression of selected genes (analyzed by ribonuclease protection assay and Northern blotting) was consistent with the frequency of their respective cDNA in the individual libraries. This porcine ovary unigene set will be useful for identifying factors and mechanisms controlling ovarian follicular development in a variety of species.

Evaluation of commercially available RNA amplification kits for RNA sequencing using very low input amounts of total RNA

This article includes supplemental data. Please visit http://www.fasebj.org to obtain this information.Multiple recent publications on RNA sequencing (RNA-seq) have demonstrated the power of next-generation sequencing technologies in whole-transcriptome analysis. Vendor-specific protocols used for RNA library construction often require at least 100 ng total RNA. However, under certain conditions, much less RNA is available for library construction. In these cases, effective transcriptome profiling requires amplification of subnanogram amounts of RNA. Several commercial RNA amplification kits are available for amplification prior to library construction for next-generation sequencing, but these kits have not been comprehensively field evaluated for accuracy and performance of RNA-seq for picogram amounts of RNA. To address this, 4 types of amplification kits were tested with 3 different concentrations, from 5 ng to 50 pg, of a commercially available RNA. Kits were tested at multiple sites to assess reproducibility and ease of use. The human total reference RNA used was spiked with a control pool of RNA molecules in order to further evaluate quantitative recovery of input material. Additional control data sets were generated from libraries constructed following polyA selection or ribosomal depletion using established kits and protocols. cDNA was collected from the different sites, and libraries were synthesized at a single site using established protocols. Sequencing runs were carried out on the Illumina platform. Numerous metrics were compared among the kits and dilutions used. Overall, no single kit appeared to meet all the challenges of small input material. However, it is encouraging that excellent data can be recovered with even the 50 pg input total RNA.

Discovery of a Novel Seminal Fluid Microbiome and Influence of Estrogen Receptor Alpha Genetic Status

Bacteria harbored in the male reproductive system may influence reproductive function and health of the male and result in developmental origins of adult health and disease (DOHaD) effects in his offspring. Such effects could be due to the seminal fluid, which is slightly basic and enriched with carbohydrates; thereby, creating an ideal habitat for microbes or a potential seminal fluid microbiome (SFM). Using wild-type (WT) and estrogen receptor-alpha (ESR1) knockout (KO) male mice, we describe a unique SFM whose inhabitants differ from gut microbes. The bacterial composition of the SFM is influenced according to whether mice have functional Esr1 genes. Propionibacterium acnes, causative agent of chronic prostatitis possibly culminating in prostate cancer, is reduced in SFM of ESR1 KO compared to WT mice (P ≤ 0.0007). In certain genetic backgrounds, WT mice show a greater incidence of prostate cancer than ESR1 KO, which may be due to increased abundance of P. acnes. Additionally, select gut microbiome residents in ESR1 KO males, such as Lachnospiraceae and Christensenellaceae, might contribute to previously identified phenotypes, especially obesity, in these mutant mice. Understanding how genetics and environmental factors influence the SFM may provide the next frontier in male reproductive disorders and possibly paternal-based DOHaD diseases.

Consumption of a high-fat diet alters the seminal fluid and gut microbiomes in male mice

Our prior work showed that a novel microbiome resides in the seminal vesicles of wild-type and oestrogen receptor α (Esr1) knock-out mice and is impacted by the presence of functional Esr1 genes. The seminal fluid microbiome (SFM) may influence the health and reproductive status of the male, along with that of his partner and offspring. A high-fat diet (HFD) alters metabolites and other factors within seminal fluid and might affect the SFM. Adult (~15 weeks old) male mice were placed for 4 weeks on a control or high-fat diet and seminal fluid and fecal samples were collected, bacterial DNA isolated and subjected to 16s rRNA sequencing. Corynebacterium spp. were elevated in the seminal fluid of HFD males; however, Acinetobacter johnsonii, Streptophyta, Ammoniphilus spp., Bacillus spp. and Propionibacterium acnes were increased in control males. Rikenellaceae was more abundant in the fecal samples from HFD males. However, Bacteroides ovatus and another Bacteroides species, Bilophila, Sutterella spp., Parabacteroides, Bifidobacterium longum, Akkermansia muciniphila and Desulfovibrio spp. were greater in control males. Thus, short-term consumption of a HFD influences the seminal fluid and fecal microbiomes, which may have important health consequence for males and developmental origins of health and disease effects in resulting offspring.

Bisphenol A (BPA) in the serum of pet dogs following short-term consumption of canned dog food and potential health consequences of exposure to BPA

Bisphenol A (BPA) is a widely present endocrine disruptor chemical found in many household items. Moreover, this chemical can bioaccumulate in various terrestrial and aquatic sources; thereby ensuring continual exposure of animals and humans. For most species, including humans, diet is considered the primary route of exposure. However, there has been little investigation whether commercial-brands of dog foods contain BPA and potential health ramifications of BPA-dietary exposure in dogs. We sought to determine BPA content within dog food, whether short-term consumption of these diets increases serum concentrations of BPA, and potential health consequences, as assessed by potential hematological, serum chemistry, cortisol, DNA methylation, and gut microbiome changes, in dogs associated with short-term dietary exposure to BPA. Fourteen healthy privately-owned dogs were used in this study. Blood and fecal samples were collected prior to dogs being placed for two-weeks on one of two diets (with one considered to be BPA-free), and blood and fecal samples were collected again. Serum/plasma samples were analyzed for chemistry and hematology profiles, cortisol concentrations, 5-methylcytosine in lymphocytes, and total BPA concentrations. Fecal samples were used for microbiome assessments. Both diets contained BPA, and after two-weeks of being on either diet, dogs had a significant increase in circulating BPA concentrations (pre-samples=0.7±0.15ng/mL, post-samples=2.2±0.15ng/mL, p<0.0001). Elevated BPA concentrations positively correlated with increased plasma bicarbonate concentrations and associated with fecal microbiome alterations. Short-term feeding of canned dog food increased circulating BPA concentrations in dogs comparable to amounts detected in humans, and greater BPA concentrations were associated with serum chemistry and microbiome changes. Dogs, who share our internal and external environments with us, are likely excellent indicators of potential human health concerns to BPA and other environmental chemicals. These findings may also have relevance to aquatic and terrestrial wildlife.