Physiology & Growth Biology Research
2009-2010
- 2010 -
Differential gene expression of ewes varying in tolerance to dietary nitrate
R.R. Cockrum1, K.J. Austin1, J.W. Kim2, J.R. Garbe3, S.C. Fahrenkrug3, J.F. Taylor2, and K.M. Cammack1
Ruminants consuming diets with increased concentrations of nitrate (NO3–) can accumulate nitrite (NO2–) in the blood, resulting in toxicity. In a previous experiment, ewes identified as highly tolerant to subacute dietary NO3– were able to consume greater amounts of NO3– than lowly tolerant ewes without exhibiting signs of toxicity. We hypothesized that highly tolerant and lowly tolerant ewes differ in their ability to metabolize NO3– and thereby differ in the expression of hepatic genes involved in NO3– metabolism. Therefore, our objective was to identify hepatic genes differentially expressed between ewes classified as lowly tolerant and highly tolerant after administration of a subacute quantity of dietary NO3–. Analysis of the Bovine Oligonucleotide Microarray data identified 100 oligonucleotides as differentially expressed (P < 0.05) between lowly tolerant and highly tolerant ewes. Functional analysis of the genes associated with these oligonucleotides revealed 2 response clusters of interest: metabolic and stress. Genes of interest within these 2 clusters (n = 17) and nonclustered genes with the greatest fold changes (FC; n = 5) were selected for validation by real-time reverse-transcription PCR. Relative expression, genomic regulation, and FC agreed between microarray and real-time reverse-transcription-PCR analyses, and FC differences (P < 0.05) between lowly tolerant and highly tolerant ewes were confirmed for 12 genes. Metabolic genes that were downregulated (P ≤ 0.032) in lowly tolerant ewes vs. highly tolerant ewes included aldehyde oxidase 1, argininosuccinate lyase, putative steroid dehydrogenase, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase1, and sterol carrier protein 2. In contrast, the metabolic gene homeobox was upregulated (P = 0.037) in lowly tolerant ewes. The glutathione peroxidase 3 and inter-α (globulin) inhibitor H4 genes in the stress response cluster were upregulated (P ≤ 0.045) in lowly tolerant ewes. Genes with the greatest FC, but did not cluster within the functional analysis included haptoglobin, which was upregulated (P = 0.024) in lowly tolerant ewes, and fatty acid desaturase 2 and thyroid hormone responsive, both of which were downregulated (P ≤ 0.019) in lowly tolerant ewes. Results from this study indicate that hepatic gene expression differs in ewes identified as lowly tolerant and highly tolerant to increased dietary NO3–.
1Department of Animal Science, University of Wyoming, Laramie
2Division of Animal Sciences, University of Missouri, Columbia
3Department of Animal Science, University of Minnesota, St. Paul
Precision genetics for complex objectives in animal agriculture
S.C. Fahrenkrug1,2,,3,4, A. Blake5, D.F. Carlson1,2,3, T. Doran6, A. Van Eenennaam7, D. Faber8, C. Galli9, Q. Gao10, P.B. Hackett2,11, N. Li12, E.A. Maga13, W.M. Muir14, J.D. Murray7,15, D. Shi4, R. Stotish15, E. Sullivan16, J.F. Taylor17, M. Walton18, M. Wheeler19, B. Whitelaw20, and B.P. Glenn21
Indirect modification of animal genomes by interspecific hybridization, cross-breeding, and selection has produced an enormous spectrum of phenotypic diversity over more than 10,000 yr of animal domestication. Using these established technologies, the farming community has successfully increased the yield and efficiency of production in most agricultural species while utilizing land resources that are often unsuitable for other agricultural purposes. Moving forward, animal well-being and agricultural sustainability are moral and economic priorities of consumers and producers alike. Therefore, these considerations will be included in any strategy designed to meet the challenges produced by global climate change and an expanding world population. Improvements in the efficiency and precision of genetic technologies will enable a timely response to meet the multifaceted food requirements of a rapidly increasing world population.
1Department of Animal Science, University of Minnesota, St. Paul
2Center for Genome Engineering, University of Minnesota, Minneapolis
3Recombinetics Inc., Minneapolis, MN
4Animal Reproduction Institute, Guangxi University, Nanning, P.R. China
5Yorktown Technologies, Austin, TX
6CSIRO Livestock Industries, Australian Animal Health Laboratory, Geelong, VIC 3220 Australia
7Department of Animal Science, University of California, Davis 95616; and
8Trans Ova Genetics, Sioux Center, IA
9Laboratorio di Tecnologie della Riproduzione, Instituto Sperimentale Italiano Lazzaro Spallanzani, Cremona, Italy
10Jiangsu Animal Husbandry and Veterinary College, Taizhou, P.R. China
11Department of Genetics and Cell Biology, University of Minnesota, Minneapolis
12State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing, P.R. China
13Department of Animal Science, Purdue University, West Lafayette, IN
14Department of Population Health and Reproduction, University of California, Davis
15Aqua Bounty Technologies, Waltham, MA
16Hematech Inc., Sioux Falls, SD
17Division of Animal Sciences, University of Missouri, Columbia, MO
18ViaGen Inc., Austin, TX
19Department of Animal Sciences, University of Illinois, Urbana-Champaign
20The Roslin Institute, The University of Edinburgh, Roslin, Midlothian, Scotland, UK
21Biotechnology Industry Organization, Washington, DC
- 2009 -
Effects of immunization against alpha-inhibin using two adjuvants on daily sperm production and hormone concentrations in ram lambs
J.L. Voge, J.B. Parker, and J.E. Wheaton
Department of Animal Science, University of Minnesota, St. Paul
Twenty-five ram lambs were immunized against alpha-inhibin peptide emulsified in Freund's adjuvant (FRA), Emulsigen (EML) containing an oligodeoxynucleotide as an immunostimulant, or adjuvant without alpha-inhibin antigen (control). Four immunizations were administered during an 85-d period, after which testes were obtained for determination of daily sperm production (DSP) and histological evaluation. alpha-Inhibin antibody (Ab) titers were 70-fold greater in lambs treated with FRA than in EML-treated ram lambs. alpha-Inhibin immunization had no effect on testes weight or on plasma concentrations of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone. Mean DSP/g tended (P=0.1) to be greater in alpha-inhibin-immunized (EML=17.6x10(6); FRA=15.8x10(6)) ram lambs than in control animals (14.4x10(6)). One of the 8 control ram lambs had an elevated DSP/g, which was a statistical outlier. Without data from this lamb, DSP/g was increased (P<0.01) in alpha-inhibin-immunized ram lambs by 28% over controls. No association was found between the titer of alpha-inhibin Ab developed and DSP/g. Histologically, the percentage of testicular area occupied by seminiferous tubules differed (P=0.01) by treatment and was greatest (82%) in EML-treated ram alpha-inhibin-immunized lambs and lowest (74%) in control animals. Percentage tubular area and DSP/g were correlated (r=0.57, P=0.003). Findings show that (1) the extent of the increase in DSP/g is not dependent on the titer of alpha-inhibin Ab; (2) the increase in DSP/g is achieved through an increase in the mass of seminiferous tubules; and (3) FRA elicits a greater alpha-inhibin Ab titer than EML containing an oligodeoxynucleotide.
Effects of immunization against alpha-inhibin anopsin in the premammillary nucleus of the avian
S.W. Kang, B. Leclerc, and M.E. El Halawani
Department of Animal Science, University of Minnesota, St. Paul
Melanopsin (cOPN4) has been proposed as an important photoreceptive
molecule regulating the avian circadian system. Previous studies in our
laboratory have shown that co-localized dopamine-melatonin (DAMEL)
neurons in the hypothalamic premammillary nucleus (PMM) are
photosensitive and exhibit circadian rhythms. This study investigates
chicken OPN4 (cOPN4) mRNA distribution in the turkey hypothalamus
and brainstem, and characterizes cOPN4 mRNA expression in PMM
DA-MEL neurons, using in situ hybridization (ISH), double-label immunocytochemistry
(ICC), double ISH/ICC, and real time-PCR. cOPN4
mRNA was found in anatomically discrete areas in or near the hypothalamus
and the brainstem, including POM (nucleus preopticus medialis),
SL (nucleus septalis lateralis), PMM and the pineal gland. Double ICC,
using tyrosine hydroxylase (TH)/cOPN4 antibodies, confirmed that the
cOPN4 protein coexisted in the DA-MEL neurons and cOPN4 mRNA
expression was verified with double ISH/ICC using cOPN4 mRNA and
TH immunoreactivity. PMM and pineal gland cOPN4 mRNA expression
levels were high during the night and low during the day, indicating
circadian rhythmicity. Stimulation with light during the dark period in
short day hens downregulated cOPN4 expression level significantly at
the avian photosensitive phase (circadian time 14 h; CT14), more so than
it did at CT8 and CT20. There was a significantly lower level of cOPN4
mRNA in PMM neurons in photorefractory hens as compared with short
and long day hens. The present study is the first to show that cOPN4 is
expressed in the DA-MEL neurons controlling seasonal reproduction and
also the first to show cOPN4 expression peaking at night as part of the
circadian rhythm. The results suggest that cOPN4 in the PMM DA-MEL
neurons in the hypothalamus might constitute an important photoreceptive
system for regulating reproductive function in the female turkey.
Photoperiodic modulation of clock gene expression in the avian premammillary nucleus
B. Leclerc1, S.W. Kang1, L.J. Mauro1, S. Kosonsiriluk1, Y. Chaiseha2, and M.E. El Halawani1
The premammillary nucleus (PMM) has been shown to contain a daily endogenous dual-oscillation in dopamine (DA)/melatonin (MEL) as well as c-fos mRNA expression that is associated with the daily photo-inducible phase of gonad growth in turkeys. In the present study, the expression of clock genes (Bmal1, Clock, Cry1, Cry2, Per2 and Per3) in the PMM was determined under short (8 : 16 h light/dark cycle) and long (16 : 8 h light/dark cycle) photoperiods relative to changes associated with the diurnal rhythm of DA and MEL. Constant darkness (0 : 24 h light/dark cycle) was used to assess the endogenous response of clock genes. In addition, light pulses were given at zeitgeber time (ZT) 8, 14 and 20 to ascertain whether clock gene expression is modulated by light pulse stimulation and therefore has a daily phase-related response. In the PMM, the temporal clock gene expression profiles were similar under short and long photoperiods, except that Per3 gene was phase-delayed by approximately 16 h under long photoperiod. In addition, Cry1 and Per3 genes were light-induced at ZT 14, the photosensitive phase for gonad recrudescence, whereas the Clock gene was repressed. Gene expression in established circadian pacemakers, the visual suprachiasmatic nucleus (vSCN) and the pineal, was also determined. Clock genes in the pineal gland were rhythmic under both photoperiods, and were not altered after light pulses at ZT 14, which suggests that pineal clock genes may not be associated with the photosensitive phase and reproductive activities. In the vSCN, clock gene expression was phase-shifted depending on the photoperiod, with apexes at night under short day length and during the day under long day length. Furthermore, light pulses at ZT 14 induced the Per2 gene, whereas it repressed the Bmal1 gene. Taken together, the changes in clock gene expression observed within the PMM were unique compared to the pineal and vSCN, and were induced by long photoperiod and light during the daily photosensitive phase; stimuli that are also documented to promote reproductive activity. These results show that Cry1 and Per3 are involved in the photic response associated with the PMM neuronal activation and are coincident with an essential circadian mechanism (photosensitive phase) controlling the reproductive neuroendocrine system.
1Department of Animal Science, University of Minnesota, St. Paul
2School of Biology, Institute of Science, Surananree University of Technology, Nakhon Ratchasima, Thailand
Potential role of low-density lipoprotein receptor-related protein (LRP)-1 and IGFBP-3 in the proliferation-suppressing actions of TGF-beta on cultured myogenic cells
E. Kamanga-Sollo,
M.S. Pampusch, M.E. White, M.R. Hathaway, and W.R. Dayton
Department of Animal Science, University of Minnesota, St. Paul
Myostatin and transforming growth factor (TGF)-beta suppress both
proliferation and differentiation of cultured myogenic cells. Recent
studies have shown that the IGF-independent actions of insulin-like
growth factor binding protein (IGFBP)-3 facilitate the proliferationsuppressing
actions of both myostatin and TGF-beta on cultured myogenic cells; however, the mechanism of this facilitation is not known.
To assess this mechanism, we have transfected L6 myogenic cells,
which do not produce detectable levels of IGFBP-3, with a construct
containing the porcine IGFBP-3 cDNA behind a constitutively active promoter. These transfected cells (tL6) constitutively express porcine
IGFBP-3. Consistent with our previous observation that IGFBP-3 facilitates
the proliferation-suppressing actions of TGF-beta, dose response
curves showed that proliferation of tL6 cells was inhibited at lower
TGF-beta concentrations than was proliferation of mock-transfected
or non-transfected L6 cells (p < 0.05). In non-muscle cells, IGFBP-3
suppresses proliferation by binding to LRP-1, suggesting that binding
to this receptor may play a role in the ability of IGFBP-3 to facilitate
the proliferation-suppressing actions of TGF-beta and myostatin on
cultured myogenic cells. To assess the role of LRP-1 in this process,
we have examined the effects of Receptor Associated Protein (RAP), a
protein which inhibits ligand binding to LRP-1, on TGF-beta-induced
suppression of proliferation of mock-transfected L6 and tL6 myogenic
cells. RAP significantly (p < 0.02) increases proliferation of TGF-betatreated
tL6 cells, which produce IGFBP-3, while having no effect on
proliferation of TGF-beta-treated mock transfected or control L6 cells,
neither of which produces IGFBP-3. These data suggest that binding
of IGFBP-3 to LRP-1 may play a role in the mechanism by which
IGFBP-3 facilitates the proliferation-suppressing actions of TGF-beta
on cultured myogenic cells.
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