Progress 10/01/03 to 09/30/08
Outputs
Using mice, we have shown that the mammalian neuroendocrine transcription factor, Nhlh2 controls both body weight and fertility/sex behavior. In this project, we have used mice to characterize Nhlh2's role in fertility, cloned the bovine NHLH2 gene (bHLH2) and begun to identify polymophisms in the bNHLH2 which could affect both fertility and body weight or fat. In essence, we have translated knowledge from mice into an agriculturally-important species, cattle. In the first year of the grant, we showed that females with a deletion of the Nhlh2 transcription factor have abnormal estrous cycles and reduced ovulation in response to exogenously administered hormones. We also confirmed that female Nhlh2 knockout mice show reduced sexual behavior (lordosis response) using either physiological or pharmacological doses of hormones. The lack of female sexual behavior, along with reduced ovulation is severely pronounced in aged females, suggesting that Nhlh2 controls the age at
which menopause occurs and that females lacking Nhlh2 progress to an earlier menopause than normal, wildtype females. In the second year of the grant, we cloned the bNHLH2 gene from Holstein cattle and used in silico comparative mapping to identify the chromosomal location of the BNHLH2 gene. The coding region of bNHLH2 is 91% homologous to human and 87% homologous to mouse Nhlh2. There is one conserved amino acid change in Holstein breed relative to Hereford (K71 => R71), which occurs in the beginning of the basic DNA binding domain. Genomic sequence from a mixed breed animal does not show this particular polymorphism. In silico mapping was used to map the bNHLH2 genomic sequence to within 100,000 bp of a known quantitative trait locus (QTL) for marbling in beef cattle. When the bovine mapping consortium published their mapping data in December 2005, it confirmed our predicted location. This past year, we have extended our analysis of the coding region of bovine NHLH2 (bNHLH2) to
Wagyu animals, which have high body fat and high marbling. Of five individual Wagyu animals sequenced, three of them had polymorphisms in bNHLH2. The amino acid changes in Wagyu bNHLH2 relative to Holstein and Hereford bNHLH2 are in the amino-terminal end of the protein, a region rich in putative motifs for phosphorylation, myristolyation and other post-translational protein modifications. We have also extended our analysis of the bNHLH2 gene to include the intron/exon structure and promoter. We have found that the intron/exon structure of bNHLH2 is conserved in both size and spacing between species. In addition, the very proximal 300 base pairs of the bovine NHLH2 gene shares 94% homology with the human NHLH2 promoter and 75% homology with mouse Nhlh2 promoter, leading us to speculate that NHLH2 may be similarly regulated in these species. Cloning and sequence analysis of bNHLH2 in each individual sample has begun and will be scanned for mutations that could affect expression of
bNHLH2. These data have been used for part of a competitive USDA CSREES grant proposal, which last year received a High Priority impact score, but was unfunded. This year the proposal is currently under review.
Impacts
Breeding programs have responded to consumer demands for meat quality for years but it is only recently that breeders have been able to use genetic markers to select for meat quality. The work in this project has translated work using mice into knowledge in an agriculturally-important species, cattle. The bNHLH2 gene is located within a quantitative trait locus for marbling in beef cattle, and we have identified several polymorphisms in the high fat Wagyu strain of cattle. Further analysis of these polymorphisms should help basic biologists as well as the cattle industry understand and correlate single nucleotide polymorphisms with body fat, marbling and fertility. These data promise to determine whether the bNHLH2 gene should be used as a genetic marker in controlled breeding programs, and have provided the basic knowledge on the expression, regulation and function of a newly identified bovine gene.
Publications
- Burnside, A.S., and Good, D.J. (2006) Genetic Diversity of Genes Involved in Body Weight Regulation in Genes, Genomes and Genomics, Vol 1, Thangadurai D, Tang W., and Pullaiah T., eds Regency Publications, New Delhi, pp74-97.
- Brennan, K.M, Vella K.R. and Good, D.J. (2006) Genetic analysis of NHLH2 and its putative role in bovine body weight control, Animal Genetics, 37, Suppl 1:24-7.
- Fox, D. L., Vella, K.R., and Good, D.J. (2006) Energy Balance Pathways Converging in the Nhlh2 Transcription Factor, in Frontiers in Bioscience, Peptides Controlling Energy Balance, invited chapter, under review
- Good, D.J. (2007) Mouse Models of Obesity and Overweight, in Sourcebook of Models for Biomedical Research, invited chapter, submission date September 15, 2006
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Progress 10/01/04 to 09/30/05
Outputs
This past year, we have focused our efforts on cloning and characterizing bNHLH2 in different breeds of beef cattle. Using PCR amplification of genomic DNA isolated from bovine embryo fibroblasts derived from Holstein cattle, and primer derived from the human and mouse NHLH2 sequence, we have cloned a 420 base pair (bp) fragment of the bovine gene. This fragment contains the entire protein coding region of bHLH2, and is nearly identical to the online sequence from the Hereford breed. Unlike human NHLH2, where to date there are no identified coding region differences, there is one conserved amino acid change in Holstein breed relative to Hereford (K71 => R71), which occurs in the beginning of the basic DNA binding domain. Genomic sequence from a mixed breed animal does not show this particular polymorphism. There are a number of conserved changes between the Holstein, Hereford and mixed breed sequences compared to the sequence from mouse, rat and human. Of all of the
mammalian Nhlh2 sequences analyzed, only bHLH2 has a proline at position 62, although avian Nhlh2 contains a proline at this position as well. Preliminary sequence analysis of bNHLH2 from Wagyu, a high fat breed indicates the presence of polymorphisms in bNHLH2 relative to sequences from Hereford, Holstein and the mixed breed animals, and consistent with a possible role for bNHLH2 in body fat control in cattle. In addition, using in silico mapping, we have shown that bNHLH2 maps within 100,000 bp of a known quantitative trait locus (QTL) for marbling in beef cattle. These data have been submitted to Animal Genetics, and were presented in a seminar given by the PI at the Functional and Integrative Genomics meeting at Iowa State in September. We have also found that the bNHLH2 protein shows 96.2 percent homology with the human and chimpanzee NHLH2 protein, and 95 percent homology with mouse or rat Nhlh2. The avian Nhlh2 paralog has only 75.5% homology and may actually represent a closer
match to the related gene NHLH1. This is true also of the Amphibian (Frog 46% homology), Fish (Pufferfish, 61.5% and Zebrafish, 61.4% homology respectively), Worm (51% homology) and Fly (63% homology) lineages. It appears that in these species there was just one ancestral gene prior to duplication and evolution into Nhlh1 and Nhlh2 genes in higher order species. In analyzing the percent conservation of amino acid sequence for a number of body weight control proteins, Nhlh2 appears to have one of the highest levels of homology among mammals. This may point to an important conservation of function for this transcription factor throughout the animal kingdom. Finally, we have shown that bNHLH2 is strongly expressed in bovine hypothalamic tissue, similar to the mouse and human expression pattern. We are currently determining the intron/exon structure of bNHLH2 and analyzing the sequence for both promoter and coding region polymorphisms in breeds with differences in marbling and body fat
content.
Impacts
We have identified NHLH2 as a central nervous system regulator of adult body weight, and shown that this gene maps near to a QTL for marbling in cattle. We are currently analyzing different breeds of cattle to determine if polymorphisms in either the coding region or promoter affect different marbling and fat content in cattle. Breeding programs have responded to consumer demands for increased meat quality for many years, but it is only recently that breeders have been able to use genetic markers to select for meat quality. Identification of polymorphisms in bNHLH2 linked to the QTL for marbling on BTA 3 may have a direct impact on using the bNHLH2 gene in future selective breeding strategies.
Publications
- Johnson, S.A., Miele, M., Marin-Bivens, C., Coyle, C.A., Fissore, R., and Good, D.J., 2004. The Nhlh2 transcription factor is required for female sexual behavior and reproductive longevity, Hormones and Behavior, 46(4):420-7 (published Nov 2004)
- Good, D.J. 2005. Using Obese Mouse Models in Research: Special considerations for IACUC members, animal care technicians and researchers. Lab Animal 34: 30-37. (published Feb 2005)
- Burnside, A.S. and Good, D.J. 2004. Mind Over Matter: Transcriptional regulation of body weight by Hypothalamic Neurons. Recent Research Developments in Molecular and Cellular Biology, 5: 23-39. (published December 2004)
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Progress 10/01/03 to 09/30/04
Outputs
Reproduction in mammals is regulated by endocrine interactions between the hypothalamus, pituitary and gonads. A thorough understanding of the molecular events controlling estrous, ovulation and sexual behavior in females is necessary for effective reproductive management of dairy and beef cattle herds. Our laboratory uses a mouse containing the targeted deletion of a basic helix-loop-helix transcription factor, Nhlh2 to study the role of this transcription factor in ferility, as it is nearly impossible to study this genetically in cattle. We have shown that female mice with a deletion of the Nhlh2 transcription factor have abnormal estrous cycles and reduced ovulation in response to exogenously administered hormones. We have also confirmed that female Nhlh2 knockout mice show reduced sexual behavior (lordosis response) using either physiological or pharmacological doses of hormones which should induce this behavior. The lack of female sexual behavior, along with
reduced ovulation is severely pronounced in aged females, suggesting that Nhlh2 controls the age at which menopause occurs and that females lacking Nhlh2 progress to an earlier menopause than normal, wildtype females. These data have been published this year in Hormones and Behavior. In extending these studies, we have data to show that Nhlh2 knockout females express altered levels of progesterone receptor in their hypothalamus during certain phases of their estrous cycle, and that this may contrubute to the irregular estrous cycles of the knockout mice. Furthermore, we have shown that mRNA for the Nhlh2 transcription factor is differentially regulated in the hypothalamus during diestrus and proestrus phases of the estrous cycle, suggesting that it not only regulates target genes during the estrous cycle, but that the Nhlh2 gene itself is a target of hormones during the estrous cycles. We have also cloned the bovine Nhlh2 gene and characterized the nucleotide and protein sequence from
both Holstein and Waygu strains of cattle. We will use the bovine Nhlh2 in order to explore the role of this gene in domesticated species such as bovine and to translate our research using our mouse model into an agriculturally-important species. As a beginning to this line of work, we have used the bovine Nhlh2 clone to analyze expression of Nhlh2 in the bovine brain, and shown that the expression pattern in cows is similar to mice, rats and humans. These data were used this summer as part of a USDA competitive grant proposal submission which is currently under review
Impacts
Successful reproduction in mammals requires the interaction of the reproductive and neuroendocrine systems. We have identified a mammalian neuroendocrine transcription factor, Nhlh2, which controls fertility and sex behavior in mice, and have translated this knowledge into an agriculturally-important species, cattle. It is hoped that these studies will contribute to a better understanding of the molecular events controlling bovine reproduction and will eventually address methods of alleviating acyclicity and reproductive inefficiency in herds.
Publications
- Johnson, S.A., Miele, M., Marin-Bivens, C., Coyle, C.A., Fissore, R., and Good, D.J., (2004) The Nhlh2 transcription factor is required for female sexual behavior and reproductive longevity, Hormones and Behavior, 46(4):420-7
- Johnson, S.A. and Good, D.J. (2004) Molecular mechanism of reduced fertility and sexual behavior in Nhlh2 knockout mice Biol. Reprod., in preparation.
- Brennan, K.A., and Good, D.J. (2004) Cloning and expression analysis of the bovine Nhlh2 gene. in preparation
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Progress 10/01/02 to 09/30/03
Outputs
New project this year, no report
Impacts
New project
Publications
- No publications reported this period
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