Progress 05/01/07 to 12/31/08
Outputs
OUTPUTS: The following activities have been carried out: 1. Isolation of bovine homologs of the human Y chromosome (chr) genes by a comparative mapping approach. Bovine testis cDNA libraries were constructed and screened with the human MSY genes as probes. 2. Isolation of bovine species-specific MSY genes by a direct testis cDNA selection approach. Bovine testis cDNAs were hybridized with a probe of the entire bovine Y chromosome (BTAY) DNA that was isolated and PCR-amplified by a micro-dissection and cloning technique. This approach allowed us to identify bovine species-specific MSY genes that are not present on human and other mammalian Y chrs. 3. Experiments were conducted to define the genomic structures and expression profiles of the BTAY genes. 4. Experiments were performed to identify MSY gene polymorphisms and their relationship with spermatogenesis and male fertility. 5. On the basis of the genes and DNA markers we developed, a radiation hybrid (RH) map of BTAY was generated. 6. Patent application. We identified a number of single nucleotide polymorphisms (SNPs) that are associated with bull fertility traits, such as non-return rate. A patent application entitled sire early selection for male fertility using SNP-haplotype of the DAZL gene (No. WO US/2008 50937) was filed. 7. Mentoring and training. In the course of this project, two post-docs, one graduate student, and five undergraduate students were trained. We developed a course in Animal Genomics, and engaged in formal classroom instruction, laboratory instruction, and practicum experiences. DISSEMINATION: Research results were presented as abstracts at a number of national and international scientific meetings, including the International Plant and Animal Genome Conference (PAG) and the International Animal Genetics Conference (ISAG), and as invited talks in China and U.S. universities and companies. We shared knowledge with cattle breeders and bull studs, especially with the leading companies, such as Select Sires, Semex and Genex, in the artificial insemination (AI) industry. We also established collaborative research projects with these companies with a purpose to evaluate the results and to transfer the knowledge into the cattle breeding and the AI industry. PARTICIPANTS: Dr. Wansheng Liu, Associate Professor, Penn State University; Dr. Yang Yang, Postdoctoral Associate, Penn State University. Partner Organizations: Select Sires, Genex and Semex provided bull semen samples for the project. Select Sires also provided gift funds for this research. TARGET AUDIENCES: a: Scientists and researchers in the fields of male fertility, animal reproduction, animal breeding and genetics, and genomics; b. Animal breeders in the dairy/beef cattle industry and A.I. industry; c. vet diagnostic laboratories/centers; d: Beef producers who still use natural service sires; e. Scientists and researchers interested in Y chromosome and molecular evolution. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
OUTCOMES: The findings from this project significantly increase our knowledge regarding the bovine Y chr in following areas: 1.There are nine genes (families), including USP9Y, DDX3Y, UTY, ZFY, EIF1AY, PRKY, SRY, TSPY and HSFY presenting on both the bovine and human Y chrs, which are essential for sex determination and male fertility. 2. The orthologs of the human DAZ and CDY, the major candidates for infertility in men, are not present on BTAY, but on autosomes. The bovine homologs of several other human MSY gene families, such as RBMY, VCY and BPY, could not be identified by a comparative mapping, indicating that genes in the MSY are poorly conserved between bovine and human. 3. The preliminary gene content of the bovine MSY region. Through a direct testis cDNA selection, we isolated a total of 34 different BTAY transcripts, including 13 known genes, 11 predicted XY-shared genes, and 10 novel transcripts. In addition to the 9 human-bovine shared MSY genes (see above), we identified four more BTAY genes that are multiple copies on MSY and also have a copy on autosomes. However, they are not present on the human and mouse Y. We also identified the bovine UBE1Y, which is present on the mouse Y, but lost in the human Y. The 11 XY-shared genes are most likely the bovine unique X-degenerate genes. The 10 novel transcripts (MSTY) are bovine species-specific as they do not have homologs on human and other mammalian Y chrs. 4. The bovine USP9Y, DDX3Y, UTY, ZFY, EIF1AY, PRKY, SRY have single copy on the Y, and also have a homolog on the X, while HSFY, TSPY, ZNF280BY and the 10 MSTY are multi-copies and Y-specific. 5. RNA alternative splicing is common to MSY-related genes, which are expressed predominantly or exclusively in the testis, suggesting that they play an essential role in spermatogenesis and fertility. 6. Sense and anti-sense transcripts are present in spermatocytes and spermotids of testis for CDYL and CDYL2, while in interstitial cells for DDX3Y. 7. The bovine CDYL gene is unique by having an additional short transcript that was not identified in humans and mice, and that would be the ancestor of the gene family. CDYL is a great model for studying exon acquisition and gene function diversification. 8. Gene (DAZL, CDYL, DDX3Y and HSFY) polymorphisms (SNPs and indels) are associated with bull fertility traits, and are potential molecular markers for male fertility selection. 9. Analysis of the ovine homologs of some bovine Y-chr genes indicated that they are highly conserved. IMPACTS: The Y-chr testis genes are the most important source for molecular study of male fertility. This study provides details of the organization and function of these Y-related genes in bovine spermatogenesis and fertility. Polymorphic markers (SNPs and indels) developed in this project allow us to haplotype high fertility, low fertility and subfertile/infertile bulls. We expect this information to significantly improve the design of new MAS strategies using Y haplotypes as an aid in selecting sires at an earlier age prior to entering a breeding program and eliminate potential genetic defects associated with reduced fertility.
Publications
- Liu, W.-S. 2008. Comparative Genomics of Y Chromosome and Male Fertility, Book chapter in Reproductive Genomics of Domestic Animals. Wiley-Blackwell Publishing, USA (Accepted).
- Liu W.-S., A. Wang, Y. Uno, D. Galtz, C.W. Beattie, and F.A. Ponce de Leon. 2007. Genomic structure and transcript variants of the bovine DAZL gene. Cytogenetics and Genome Research 116, 65-71.
- Liu, W.-S. and F.A. Ponce de Leon. 2007. Mapping of the Bovine Y Chromosome. Electronic Journal of Biology 3 (1), 5-12.
- Liu, W.-S. 2007. Sire early selection for male fertility using SNP-haplotype of the DAZL gene. Patent Cooperation Treaty application (WO US/2008 50937). P1-45.
- Wang, A., F.A. Ponce de Leon, and W.-S. Liu. 2007. The bovine CDYL gene family: cloning, mapping and expression analysis. Conference Abstract, PAG-XV. P530.
- Liu, W.-S. and F.A. Ponce de Leon. 2006. Characterization and Mapping of the Bovine DAZL Gene. Conference Abstract, PAG-XIV. P541.
- Landrito E., A. Wang, and W.-S. Liu. 2006. Cloning and sequencing of the bovine DBY and USP9X genes. Dept of Biochem. & Mol. Bio. University of Nevada, Reno. Poster W3. http://www.ag.unr.edu/Condit/bch121/SeniorPapersW3.html
- Ponce de Leon, F.A., W.-S. Liu, and C.M Carpio. 2006. Understanding the organization of BTAY and male fertility genes. Conference Abstract, the 14th North American Colloquium on Domestic Animal Cytogenetics and Gene Mapping. July 3-7, 2005. Puerto Vallarta, Jalisco, Mexico. Chromosome Research 14, 219.
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Progress 05/01/07 to 04/30/08
Outputs
OUTPUTS: 1. We have analyzed the genomic structures and transcript variants of three more bovine Y-chr genes/families: DDX3Y, HSFY and EIf1AY. We found two transcripts for bDDX3Y, which correspond to the long and short transcripts of the human DDX3Y. The two transcripts are identical except for a 3 bp insertion and an expanded 3'UTR in bDDX3Y-L. The bDDX3Y-S encodes a peptide of 660 aa, while the bDDX3Y-L, a 661 aa, as a result of the insertion of a serine in the bDDX3Y-L peptide. Unlike the human DDX3Y-L that is expressed ubiquitously, both transcripts of bDDX3Y are expressed solely in the bovine testis. In situ hybridization (ISH) with cRNA probes on the testicular sections revealed that bDDX3Y transcripts were expressed in the interstitial cells. We have identified six transcripts of the bHSFY gene from an adult testis, which can be classified into two groups. Group I contains 3 transcripts with a different size of 3'UTR that encode a peptide of 207 aa, whereas group II contains the remaining 3 transcripts that encode a 417 aa. One of the group II transcripts shows a deletion of 9 bp, resulting in an isoform of 414 aa. Our preliminary data indicated that multi-copies of the bHSFY gene are present on the bovine Y chromosome. We have also obtained the full-length cDNA of the bEIF1AY gene, which is 717 bp in length. It has 7 exons and 6 introns and encodes a peptide of 143 aa. 2. We have finished the characterization of the ovine homologs of the bDAZL and bCDYL genes. The ovine and bovine genes are highly conserved with a 95-99% similarity at mRNA and protein levels. 3. We found that the bCDYL gene is very unique and more complex in terms of gene transcription when compared to the human CDY/CDYL and mouse Cdyl gene. Two types of CDYL transcripts have been reported. Type I is considered the ancestral type, while type II emerged in the course of evolution with the acquisition of new exons in the 5' region and utilization of a different promoter. We have identified at least six bCDYL transcript variants. Among these, variant 1 was structurally similar to human CDYL variant 1. The bCDYL variant 2a was similar, at the mRNA level, to human variant 2; however, at the protein level, the deduced bCDYL isoform b was not like human isoform b, but similar to human isoform a1. The bCDYL variant 2b was similar to human isoform b. The bCDYL variant 3 is very similar to the human variant 3, whereas the bCDYL variant 4a and 4b are first reported in the bovine. Gene structure and promoter analyses indicated that bCDYL variant 4a and 4b may use a different promoter and form a new type (Type III), which could be the proto-ancenstral type in mammals. Therefore, Type I and II probably evolved on the basis of Type III. 4. We have identified a total of ~80 SNPs from the bDAZL, bCDYL, bDDX3Y, bHSFY and bEIF1AY genes, which are under validation and will be genotyped among a group of ~480 bulls with different performance in fertility. We have also identified several indels for the bDDX3Yand bHSFY genes. A patent has been filed (PCT WO US/2008 50937) for our discoveries in bDAZL SNPs (and haplotypes) and their association with male fertility trait (non-return rate). PARTICIPANTS: Dr. Liu, W.-S., Project Director; Dr. Ponce de Leon, F.A., Collaborator; Dr. Wang, A., Post-doc Research Associate; Dr. Yang, Y., Post-doc Research Associate; Dr. Yasue, H., Collaborator; Dr. Lin, L., Collaborator; Dr. Takashima, M.,Collaborator. TARGET AUDIENCES: a: Animal breeders in the dairy/beef cattle industry and A.I. industry; b. vet diagnostic laboratories/centers; c: beef producers who still use natural service sires; and c. scientists and researchers in the fields of animal science, vet science, animal reproduction, animal breeding, genetics, genomics, molecular biology and molecular evolution. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
One of the critical components of the reproductive process is bull fertility. In beef industry, it was estimated that as high as 18% of beef bulls used in natural service are reproductive deficient. Therefore, subfertility/infertility is a significant problem. However, this problem has not been studied at a molecular level because of the lack of molecular genetic markers and diagnostic tools, which have prevented the identification of high fertility, subfertile or infertile sires at an early age. Since very few autosomal genes that are associated with measures of germ cell defects in mammals have been identified, the Y-chromosome "testis genes" become the most important source for molecular study of male fertility/infertility. This study provides details of the organization and function of these Y-related genes in bovine spermatogenesis and fertility and additional markers (SNPs) that allow us to haplotype high fertility, low fertility and subfertile/infertile bulls. We expect this information to significantly improve the design of new MAS strategies using Y haplotypes as an aid in selecting sires at an earlier age prior to entering a breeding program and eliminate potential genetic defects associated with reduced fertility.
Publications
- Wang, A., H. Yasue, L. Lin, M. Takashima, F.A. Ponce de Leon, and W.-S. Liu. 2008. Molecular characterization of the bovine Chromodomain Y-Like genes. Animal Genetics. 39(3):207-216.
- Liu, W.-S., A. Wang, Y. Yang, E. Landrito, and H. Yasue. 2008. Molecular characterization of the bovine DDX3Y gene. Manuscript in preparation. ASAS & ADSA, Indianapolis, IN. July 7-11, 2008. Indianapolis, IN. P64, T34 (Abstract)
- Liu, W.-S., A. Wang, and H. Zhang. 2008. Polymorphisms of the bovine DAZL gene are associated with male fertility. PAG-XVI, San Diego, CA. Jan 12-16, 2008. P174 (Abstract)
- Liu, W.-S., A. Wang, Y. Yang, E. Landrito, and H. Yasue. 2008. Characterization of two bovine Y chromosome genes, DBY and HSFY. ISAG, Amsterdam, The Netherlands, July 19-24, 2008. P2094 (Abstract)
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