Progress 10/01/11 to 09/30/16
Outputs
Target Audience:Scientists in the fields of reproduction, male and female fertility, animal breeding and genetics, comparative genomics and molecular evolution; AI companies and cattlemen in the dairy and beef industry. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has provided training opportunity for 1 undergraduate student, 1 visiting post-doc and 1 visiting scholar. How have the results been disseminated to communities of interest?The results have been disseminated to communities of interest mainly through the following two ways: publications in the scientific communities, and collaboration with the AI companies and pure breed associations in the dairy industry. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
1. Subcellular localization of the PRAMEY protein in bovine spermiogenesis We reported previously the initial characterization of the PRAMRY protein by Western blot and immunofluorescence using a custom-made anti-PRAMEY antibody. We found that PRAMEY is expressed in the testis during/after puberty and predominantly in the acrosome of spermatids and mature spermatozoa. To further study the subcellular localization of PRAMEY, we performed immunogold electron microscopy (IEM) with mature bull testicular samples collected from a local slaughter house. The samples were fixed in paraformalclehyde, processed in a Leica automatic freeze-substitution system and then embedded in Lowicryl HM20 at low temperature. Cross-sections were stained with the anti-PRAMEY antibody and a secondary antibody conjuncted with gold particle. The sections on EM grids were observed under a transmission electron microscope. The results showed that the bovine PRAMEY protein was expressed in all steps of spermatids and acrosome and flagellum of spermatozoa. The enrichment of gold particles was observed in several cell organelles, including Golgi vesicles, chromatoid body (CB), acrosomal granule, centrioles and flagellums at certain steps. In the Golgi phase (steps 1-3), clusters of labeling were first seen in small vesicles that contain a CB-like structure. The protein was consistently expressed during the formation of the CB. Clusters of gold labeling were also seen in the nucleus. In the cap/acrosome phase (steps 4-12), significant enrichment of gold particles was observed in the CB before the formation of the acrosomal granule, and during migration of the CB toward the caudal pole of nucleus. Along the formation of acrosome, significant gold labeling was observed constantly within the matrix of the acrosomal granule, where the gold particles formed a unique but unknown pattern that differed from the clusters of labeling observed from other organelles. The gold labeling was then seen clearly after flattening of the acrosomal granule, particularly in the matrix of acrosome. The PRAMEY labeling was observed in both proximal and distal centrioles, mainly in those centrioles that have migrated into the caudal pole of nucleus. Clusters of labeling were also observed in the space between the proximal centriole and nucleus, as well as microtubules in the distal centriole during the formation of flagellum. In the maturation phase (step 13-14), the gold particles were primarily located in the acrosomal matrix and the out layer of flagellum. Our results strongly suggest that the bovine PRAMEY plays a functional role during spermiogenesis. 2. Male fertility evaluation with a custom-made 384-SNP chip in cattle. We reported in the last two years the generation and validation of a custom-made bovine 384-SNP chip with the Illumina VeraCode GoldenGate technology. The chip has been validated among 935 AI bulls with detailed phenotypic records in sperm morphology 1-3 (Morph1, 2, 3) and sperm motility. The genotypic data were analyzed using the Illumina Genome Studio software. Associations of genotypes with sperm quality traits were calculated using linear model procedures in R: Yi = μ + Gi + ei. By a breed-combined approach, a total of 37 significant SNPs (p < 0.05) were identified across all traits with corresponding effects and false discovery rate (FDR)-corrected p-values. Twenty-three significant SNPs for Morph1, 8 for Morph2, 22 for Morph3, and 14 for sperm motility were identified. When individual breed approach was applied, a total of 51 significant SNPs (p < 0.05) were identified, 16 of which overlapped with the markers identified from the breed-combined approach, the remaining 35 were new makers. Taken together, the breed-combined and individual breed approaches discovered a total of 72 SNPs from 65 candidate genes that have been confirmed to be involved in spermatogenesis and male fertility. The potential of using this chip for male fertility selection at an early age in cattle is under evaluation. 3. Determine the functional role of PRAME in spermiogenesis using a Prame-knock-out (KO) mouse model. We have purchased the Prame first allele KO mice for generating a conditional KO (cKO) model for the spermiogenesis study. We have also working to generate a mouse Pramel1 cKO. By collaboration with Dr. Jon Oatley, we are generating a direct KO mouse model for Pramel1 by CRISPR/Cas9.
Publications
- Type:
Books
Status:
Published
Year Published:
2016
Citation:
Zeng, W., Pan C., Liu, W.-S. (2016) Regulatory role of non-coding RNAs during spermatogenesis. Spermatogenesis: Molecular Mechanisms, Regulation and Biological Perspectives. Ed: Robison, G. Nova Science Publishers, Inc. New York. P31-70.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Lu, C., Zhao, Y.-Q., Ning, G., Liu, W.-S. (2016) The bovine PRAMEY is involved in spermiogenesis as revealed by TEM. Conference Abstract, the 47th Annual Meeting of the Society for the Study of Reproduction (SSR2016), July 16-20, 2016. San Diego, CA, USA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Liu, W.-S., Yue, X.-P., Chang, T.-C., Adams, H., Krieger, K.B. (2016) Male fertility evaluation by a candidate gene approach. Conference Abstract, ISAG 2016, July 22-27, Salt Lake City, UT, USA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Pate, J.L., Maalouf, S.A., Hughes, C., Liu, W.S. (2016) Micromanaging the CL: Regulation of transitional states in the corpus luteum by microRNA Conference Abstract, the 47th Annual Meeting of the Society for the Study of Reproduction (SSR2016), July 16-20, 2016. San Diego, CA, USA.
|
Progress 10/01/14 to 09/30/15
Outputs
Target Audience:Scientists in the fields of reproduction, male and female fertility, animal breeding and genetics, comparative genomics and molecular evolution; AI companies and cattlemen in the dairy and beef industry. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has provided training opportunity for 1 post-doc and 1 visiting scholar. How have the results been disseminated to communities of interest?The results have been disseminated to communities of interest mainly through the following two ways: publications in the scientific communities, and collaboration with the AI companies and pure breed associations in the dairy industry. What do you plan to do during the next reporting period to accomplish the goals?1. Continue to work on the Y chromosome variations (CNVs and SNPs) and male fertility in cattle and other ruminants. 2. Further characterization of the bovine PRAMEY protein during spermatogenesis. 3. Develop a Pramel1conditional knock-out (KO) mouse model for the functional study of PRAMEY/PRAME in spermatogenesis and fertilization.
Impacts
What was accomplished under these goals?
Impacts: Our work on the PRAMEY provides insights into the molecular mechanism underlying the functions of PRAMEY during spermiogenesis, which will advance the field by adding understanding of the complexity of the development of male germ cells. Our findings Holstein Y study provide a start point for investigating the impact of the extremely limited number of Y-lineages on male reproduction and other traits important for the future of the Holstein breed. The custom-made 384-SNP chip has been confirmed to be valuable for bull semen quality and fertility evaluation in dairy cattle, with a potential to be commercialized for bull fertility selection in the future. The reference methylome we discovered for high producing Holstein cattle provides a resource to more fully evaluate relationships between variation in DNA methylation and phenotype. Unraveling the interactions of DNA methylation with variation in species that have undergone intense artificial selection can provide insights into the role that DNA methylation plays in populations subject to natural and artificial selection. Results: 1. Subcellular localization of the PRAMEY protein in bovine spermiogenesis. Adult testis tissues and mature epididymal spermatozoa were collected from a local abattoir and used in the IEM experiments. The results indicated that the immunogold particles of PRAMEY are restricted to the ground substance/matrix of the pre-acrosomal granule (or the acrosome) without evidence ofany membrane association in the Stage I round spermatids and the Stage II round spermatids when the spermatids just begin to elongate. It appears that the gold particles were nonrandomly distributed in the pre-acrosomal granule and were associated with the matrix structure. Compared to the very low (almost no) background labels across the cell organelle, gold particles were also found in regions around Golgi complex. Along the formation and expansion of the acrosomal vesicle (spreading over the nucleus) during the differentiation of the spermatids, enriched gold particles were distributed in the acrosomal matrix at the top of the head (near the apical ridge) and a tendency of association with the inner acrosomal membrane (IAM) on the bottom of the head was observed in the elongated spermatids and mature spermatozoa. Our preliminary IEM data suggests that the PRAMEY protein is localized in Golgi complex, the pre-acrosomal granule that is enveloped in the Golgi vesicle, and acrosomal matrix, signifying a functional role of PRAMEY in acrosome formation in spermiogenesis. 2. Analaysis of miRNA of the corpus luteum (CL) at maternal recognition of pregnancy. We have been continued to work on those differentially expressed miRNAs between CL of pregnant vs. cyclic animals we reported last year. We further characterized the deep-seq reads count and the predicted target genes of the differentially expressed miRNA. For details please see Dr. Joy Pate's 2015 report. 3. Male fertility evaluation with a custom-made 384-SNP chip in cattle. We reported the development and evaluation of a custom-made 384-SNP chip for bull fertility analysis two years ago. In 2014-2015, we genotyped a total of 935 AI bulls from the Genex population, including 702 Holsteins, 114 Jersey and 119 Angus. Associations of genotypes with sperm quality traits were calculated using linear model procedures in R with two approaches, combined-breed and individual breed. We have finished the combined-breed analysis and identified 37 significant SNPs (p < 0.05) across all traits with corresponding effects and false discovery rate (FDR)-corrected p-values. No significant SNPs were identified for sperm morphology 1 (Morph1). These 37 significant SNPs were from 35 genes that map to 19 different autosomes in the bovine genome. The Venn diagram analysis indicated that Morph2 appears to have a larger impact on both Total Morph and Total Mot (motility) than Morph1 and Morph3. 4. A limited number of Y chromosome lineages present in North American Holsteins. The objective of this study was to investigate effective population size of the Holstein Y-chromosomes and the effects of the limited Y-chromosome lineages on male reproduction. Paternal pedigree of 62,897 Holstein bulls born during 1950-2013 in North America and 220,872 bulls registered in Interbull were analyzed. The results indicated that the number of Y-chromosome lineages in Holsteins has undergone a dramatic decrease during 1950-2013 as a consequence of artificial selection and the application of artificial insemination (AI) technology. All current Holstein AI bulls in North America are the descendants of only two ancestors born in 1880. These two ancestral Y-lineages are continued through three dominant pedigrees from the 1960s, namely Pawnee Farm Arlinda Chief, Round Oak Rag Apple Elevation, and Penstate Ivanhoe Star, with a contribution of 48.78%, 51.06% and 0.16% to the population in the 2010s, respectively. The genetic variations in the two ancestral Y-lineages were evaluated among 257 bulls by determining the copy number variations (CNVs) of three Y-linked gene families, PRAMEY, HSFY and ZNF280BY. No significant difference was found between the two ancestral Y-lineages although large CNVs were observed within each lineage. 5. Sheep and goat Y chromosome analysis. We reported the build of a sheep Y chromosome cotig for the pseudoautosomal region (PAR) in 2013. We have been continued to work on the ovine and caprine Y chromosome by further analyze the PAR and Y-specific genes through transcriptome analysis of a sheep and goat testis RNA-seq data. 6. Differential expression analysis of placentae from overfed/obese ewes and lean ewes fed only to requirements. In several mammalian species including sheep and humans, obesity during pregnancy predisposes offspring to obesity, insulin resistance, and cardiovascular disease. This study investigated how maternal diet alters gene expression in the placenta, the organ responsible for providing maternal nutrients to the developing fetus. The maternal nutrient environment to which the placenta is exposed determines maternal:fetal nutrient exchange and subsequent programming of gene expression patterns in the conceptus. Deviation from a healthy maternal diet results in altered offspring phenotype and leads to predisposition to obesity and other diseases. Differential expression analysis was performed on placental tissue from pregnant ewes fed either a control (100% of requirements) or an obesogenic (150%) diet. We performed pathway and enrichment analysis on the differentially expressed genes and identified individual genes and broader pathways that are candidates for further investigation.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2015
Citation:
Maalouf, S.W., Liu, W.-S., Pate, J.L. (2015) MicroRNA in ovarian function. Cell and Tissue Research (in press).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Liu, W.-S., Dechow, C., Yue, X.-P., (2015) The Holstein Y-chromosome: Only two Y-lineages survived in North America. Conference Abstract, PAG-XXIII, January 10-14, San Diego, CA. P276.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Dechow, C., Liu, W.-S. (2015) Genome Wide Methylation Patterns in Holstein Leukocytes. Conference Abstract, PAGXXIII,January 10-14, San Diego, CA. W136.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Guida, S. M., Mudge, J., Cameron, C. T., Lindquist , I. E., Beattie, C.W., Liu, W.-S., Ford, S. P. (2015) Differential
Expression Analysis of Placentae from Overfed/Obese Ewes and Lean Ewes Fed Only to Requirements. Conference Abstract, PAG-XXIII, January 10-14, San Diego, CA. P170.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Liu, W.-S., Zhao, Y.Q., Ning, G. (2015) Subcellular localization of the PRAMEY protein in bovine spermiogenesis. Conference Abstract, the 48th Annual Meeting of the Society for the Study of Reproduction (SSR2015), June 18-22, Puerto Rico. P541.
|
Progress 10/01/13 to 09/30/14
Outputs
Target Audience: Scientists in the fields of reproduction, male and female fertility, animal breeding and genetics, comparative genomics and molecular evolution. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? This project has provided training opportunity for 1 graduate student, 1 undergraduate student, 1 post-doc and 1 visiting scholar. How have the results been disseminated to communities of interest? The results have been disseminated to communities of interest mainly through the following two ways: publications in the scientific communities, and collaboration with the AI companies and pure breed associations in the dairy industry. What do you plan to do during the next reporting period to accomplish the goals? 1. Further characterization of the bovine PRAMEY protein during spermatogenesis. 2. Improve the ovine Y chromosome map. 3. Continue to work on the Y chromosome variations (CNVs and SNPs) and male fertility in cattle and other ruminants.
Impacts
What was accomplished under these goals?
1. Characterization of the bovine PRAMEY gene during spermatogenesis. Preferentially expressed antigen in melanoma (PRAME) has been extensively studied in cancer biology and is believed to play a regulatory role in cancer cells. The function of PRAME during spermatogenesis has not been well characterized. In the previous year (2012-2013), we reported that the mouse PRAMEL1 (on autosome) play a role in acrosome biogenesis and sperm motility. In order to study the functional role of the Y-linked gene, PRAMRY, during spermatogenesis in cattle, we have generated and validated a custom peptide-specific antibody for the bovine PRAMEY. Western blot analysis indicated that the bovine PRAMEY is expressed in testis starting at 8 months of age when the animal reaches puberty. Immunofluorescent staining revealed that the PRAMEY protein was expressed in the acrosome of spermatids, as well as the acrosome and flagellum of spermatozoa. Immunogold electron microscopy further revealed that PRAMEY was firstly seen in the acrosomal granule of round spermatids, migrated with the content of acrosomal granule during spermiogenesis, and was finally present in the acrosome matrix of mature spermatozoa. To identify the PRAMEY interactive protein(s), co-immunoprecipitation (co-IP) was performed using the PPP1CC and PRAMEY antibodies in bovine spermatozoa. The results clearly indicated that PRAMEY interacted with PPP1CC in caput and caudal epididymal sperm, suggesting a fundamental role of PRAMEY in male fertility because PPP1CC is a testis/spermatozoa specific phosphatase and a key component for regulation of spermatozoa motility and male fertility. 2. Copy number variation (CNV) of HSFY and ZNF280BY in cattle. We investigated CNVs of the bovine HSFY and ZNF280BY in a total of 460 bulls from 15 breeds. We observed variations for both gene families within and between cattle breeds. The median copy number (MCN) of HSFY among all bulls was 197, ranging from 21 to 308. The MCN of ZNF280BY was 236, varying from 28 to 380. Furthermore, bulls in the Bos taurus (BTA) lineage had a significantly higher MCN (202) of HSFY than bulls in the Bos indicus (BIN) lineage (178), while taurine bulls had a significantly lower MCN (231) of ZNF280BY than indicine bulls (284). In addition, the CN of ZNF280BY was positively correlated to that of HSFY on the bovine Y chromosome. Association analysis revealed that the CNVs of both HSFY and ZNF280BY were correlated negatively with testis size, while positively with sire conception rate. We concluded that the bovine HSFY and ZNF280BY gene families have extensively expanded on the Y chromosome during evolution, and the CNVs of HSFY and ZNF280BY may serve as valuable makers for male fertility selection in cattle. 3. Analaysis of MicroRNA (miRNA) of the corpus luteum at maternal recognition of pregnancy. The corpus luteum (CL) is a transitory endocrine gland; the dynamic nature of the CL makes it candidate for regulation by miRNA. Rescue of the CL from luteolysis is essential for the maintenance of pregnancy in all eutherian mammals. Using next generation sequencing, we profiled miRNA expression in the bovine CL during maternal recognition of pregnancy. We identified 590 luteal miRNA, of which 544 were known and 46 were novel miRNAs. Fifteen (including 3 novel) miRNAs were differentially expressed between CL of pregnant vs. cyclic animals. Target analysis of the differentially expressed miRNA resulted in genes involved in regulating apoptosis and immune response, providing evidence that miRNAs regulate the intracellular pathways that lead to either luteal regression or survival. 4. Male fertility evaluation with a custom-made 384-SNP chip in cattle. Infertility or subfertility is a common problem in humans and other mammals. Approximately 10-15% of human couples world-wide are affected by reduced rates of fertility, with 30-50% of cases being due to male factors. In the dairy industry, a variation of up to 25% of non-return rate is evident within a population of AI bulls that meet the normal commercially acceptable standards. In the beef industry, 18-30% of beef bulls used in natural service are reproductively deficient. Recent progress in genome-wide association studies (GWAS) with SNP markers makes it possible to design genetic diagnostic assays for bull fertility selection. Here we report the generation and validation of a custom-made bovine 384-SNP chip with the Illumina VeraCode GoldenGate technology. This chip contains 237 autosomal, 4 X-linked, and 143 Y-linked SNPs, from 192 autosomal, 4 X-linked and 17 Y-linked genes/families that have been confirmed to play a role in spermatogenesis, sperm function, and/or semen quality. The chip was validated among 520 Holstein AI bulls with detailed phenotypic records. The genotypic data were analyzed using the Illumina Genome Studio software and the association study was conducted with the ASReml package. Our preliminary results revealed that 123 SNPs have minor allele frequency (MAF)>0.05. Of these, 118 SNPs were found to be significantly associated with conception rate, sperm production EBV, sperm motility, and sperm morphology (p<0.05). Linkage disequilibrium and haplotype analysis are currently under way. The potential of using this chip for male fertility selection in cattle at an early age will be evaluated.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Yue, X.-P. , Dechow, C. D., Chang, T.-C., DeJarnette, J. M., Marshall, C. E., Lei, C. Z., Liu, W.-S. (2014) Copy number variations of the extensively amplified Y-linked genes, HSFY and ZNF280BY, in cattle and their association with male reproductive traits in Holstein bulls. BMC Genomics 15(1),113.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Maalouf, S.W., Liu, W.-S., Albert, I., Pate, J.L. (2014) Regulating life or death: Potential role of microRNA in rescue of the corpus luteum. Molecular and Cellular Endocrinology. Available online on Oct 14, 2014. DOI: 10.1016/j.mce.2014.10.005.
- Type:
Book Chapters
Status:
Published
Year Published:
2014
Citation:
Liu, W.-S., Chang, T.-C. (2014) Y chromosome-linked genes implicated in spermatogenesis in cattle. Reproduction in Domestic Ruminants VIII. Ed: Jennifer Juengel, Akio Miyamoto, Christopher Price, Larry Raynolds, Mike Smith, and Robert Webb. Context Products Ltd., England. P239-255. DOI: 10.13140/2.1.1392.9922.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2014
Citation:
Yue, X.-P. , Dechow, C.D., Liu, W.-S. (2014) A limited number of Y chromosome lineages present in North American Holsteins. The manuscript was submitted to J. Dairy Sci. on July 9th, and is under peer review.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Liu, Z.-H., Liu, W.-S., Ma, Y., Yue, X.P., Xiao, H.M., Li, J.-Q. (2014) Transcriptome comparison between the pubertal and adult testis in goat. Conference Abstract, the 10th World Congress on Genetics Applied to Livestock Production (WCGALP14), August 17-22, Vancouver, Canada. P915.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Yue, X.-P., Dechow, C. D., Chang, T.-C., DeJarnette, J. M., Marshall, C. E., Lei, C. Z., Liu, W.-S. (2014) CNVs of two Y-linked genes, HSFY and ZNF280BY, in cattle and their association with male fertility in Holstein sires. Conference Abstract, International Plant and Animal Genome Conference (PAG)-XXII, January 11-15, San Diego, CA. P548.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Maalouf, S.W., Liu, W.-S., Albert, I., Pate, J.L. (2014) MicroRNA of the corpus luteum at maternal recognition of pregnancy. Conference Abstract, SSR2014, July 19-23, Grand Rapids, MI. P551.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Zhao, Y.Q., Ning, G., Ocon-Grove, O.M., Diaz, F., Liu, W.-S. (2014) PRAMEY is a novel male germ cell-specific protein involved in acrosome biogenesis and blockage to polyspermy during fertilization in cattle. Conference Abstract, the 47th Annual Meeting of the Society for the Study of Reproduction (SSR2014), July 19-23, Grand Rapids, MI. No. 12.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2104
Citation:
Liu, W.-S. (2014) What have we learned from the bovine Y Chromosome? Conference Abstract, the 34th International Society for Animal Genetics Conference (ISAG2014), July 27-August 1, Xian, PR China.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Liu, W.-S., Chang, T.-C. (2014) Y chromosome linked genes implicated in spermatogenesis in cattle. Conference Abstract, the 9th International Ruminant Reproduction Symposium (IRRS2014), August 25-29, Obihiro, Japan. P23.
|
Progress 10/01/12 to 09/30/13
Outputs
Target Audience: Scientists in the fields of reproduction, male fertility, animal breeding and genetics, comparative genomics and molecular evolution. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? This project has provided training opportunity for 2 graduate students, 1 undergraduate students and 1 post-doc. How have the results been disseminated to communities of interest? The results of studies conducted during the past year demonstrate that the PRAME/PRAMEY gene family is essential for spermatogenesis and male fertility. Characterization of the bovine and ovine Y chromosome sequence, organization, and gene content provides not only important comparative mapping information for studying Y chromosome biology and evolution, but also male-specific genetic markers that will allow us to identify high and low fertility bulls in a sire selection program. What do you plan to do during the next reporting period to accomplish the goals? Work planned for the next year: 1. Further characterize the novel BTAY transcripts obtained from the testis RNA-seq data; 2. Functionally characterize the bovine PRAMEY genes; 3. Improve the OARY contig map; 4. Investigate CNVs of other BTAY-linked genes (such as HSFY, ZNF280BY, etc.).
Impacts
What was accomplished under these goals?
1. Transcriptome analysis of the bovine Y chromosome (BTAY). We have continued our research on the transcriptome of BTAY and published the results in PNAS (Chang et al. 2013) (also see report in the NRSP-8 project). We identified a total of 386 novel transcripts in addition to the 17 known bovine MSY genes (families). These novel transcripts include one gene family encoding for EGLY, ten potential protein-coding genes, termed Bovid-specific Transcript, Y-linked 1-10 (BTY1-10), and 375 non-coding RNAs (ncRNAs). Temporal and spatial expression analyses revealed that the majority of the novel BTYs and ncRNAs were expressed predominantly in testes and were up-regulated during testis development. 2. Characterization of the Pramel1 gene during spermatogenesis in the mouse. The temporal and spatial expression of the mouse Pramel1 gene, and the cellular localization of the PRAMEL1 protein during the mouse spermatogenesis were determined. The mouse Pramel1 was expressed in testis only. The mRNA and protein expression level was low in the newborn testes, and gradually increased from 1- to 3-week-old testes, and then remained constant after three weeks of age. PRAMEL1 was localized in the cytoplasm of spermatocytes and the acrosomal region of round, elongating and elongated spermatids. Furthermore, the protein localization patterns of PRAMEL1 were coordinated with morphological alterations during acrosome formation in spermatids, and were significantly different in connecting piece, middle piece and principal piece of the flagellum between testicular and epididymal spermatozoa. Collectively, our results suggest that PRAMEL1 may play a role in acrosome biogenesis and sperm motility. 3. Copy number variation (CNV) of PRAMEY and male reproduction in cattle. A quantitative real-time PCR method was applied to measure the copy number (CN) of PRAMEY among 460 bulls using a Y-linked single copy gene, DDX3Y, as a reference. The results revealed that the median CN of PRAMEY was 13, ranging from 2 to 31. Significant variations in the PRAMEY CN were observed among 15 breeds investigated. Holstein bulls had the lowest median CN (12), whereas Limousin bulls possessed the highest median CN (26). Association analysis revealed that the PRAMEY CN was correlated negatively with scrotal circumference (SC), relative scrotal circumference (RLSC), percentage of normal sperm (PNS), and non-return rate (NRR), but had no significant correlation with post thaw motility (PTM), incubated motility (IM), percentage of intact acrosome (PIA), sire conception rate (SCR) and relative breeding efficiency (RBE). 4. Build a contig map for the ovine Y chromosome. We characterized the ovine MSY (oMSY) by a combination of whole genome shotgun sequence (WGS) and BAC end sequence (BES). Alignment of the ovine BESs against the bMSY identified 605 Y chromosome BACs with one or both ends matched, of which 60 mapped in the X-degenerate and 545 in the ampliconic region. The X-degenerate region contains genes well conserved with the bovine orthologs. The ampliconic region also contains a similar genetic composition to the bMSY, though its precise genomic organization requires further testing.
Publications
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2013
Citation:
Yue, X.-P. , Chang, T.-C., DeJarnette, J. M., Marshall, C. E., Lei, C. Z., Liu, W.-S. 2013. Copy number variation of PRAMEY across breeds and its association with male fertility in Holstein sires. J Dairy Science (in press).
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Chang, T.-C., Yang, Y., Retzel, E., Liu, W.-S. 2013. Male-specific region of the bovine Y chromosome is gene rich with a high transcriptomic activity in testis development. PNAS 110(30), 12373-12378.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Mistry, B.V., Chang, T.-C., Yasue, H., Chiba, M., Zhao, Y.-Q., Oatley, J., Diaz, F., Liu, W.-S. 2013. Differential expression of PRAMEL1, a cancer/testis antigen, during spermiogenesis in the mouse. PLoS ONE 8, e60611.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Vallimont, J.E., Dechow, C.D., Daubert, J.M., Dekleva, M.W., Bl�m, J., Barlieb, C.M., Liu, W.-S, Varga, G.A., Heinrichs, A.J., Baumrucker C.R. 2013. Feed utilization and its associations with fertility and productive life in 11 Commercial Pennsylvania Tie Stalls. J. Dairy Sci. 96, 1251-4.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Zhao, Y.-Q., Mistry, B.V., Diaz, F., Liu, W.-S. 2013. PRAME is involved in spermatogenesis. Conference Abstract, the 46th Annual Meeting of the Society for the Study of Reproduction (SSR2012), July 22-26. Montreal, Canada. P457.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Yue, X.-P., Chang, T.-C., Liu, W.-S. 2013. Copy Number Variation and Association Studies of PRAMEY in Cattle. Conference Abstract, PAG-XXI, Jan 11-16. San Diego, CA. P561.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Liu, W.-S., Chang, T.-C., Yang, Y., Retzel, E. 2013. Novel Transcripts Identified in the Bovine Y Chromosome. Conference Abstract, PAG-XXI, Jan 11-16. San Diego, CA. P542.
Yue, X.-P., Chang, T.-C., Liu, W.-S. (2013) Copy Number Variation and Association Studies of PRAMEY in Cattle. Conference Abstract, PAG-XXI, Jan 11-16, 2013. San Diego, CA. P561.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Ingrid E. Lindquist, I.E., Cameron, C.T., Mudge, J., Retzel, E., Beattie, C.W., Liu, W.-S., Ford, S.P. 2013. De novo Transcriptome Assembly and Analysis of Placental Tissue from Undernourished, Control Fed and Obese Pregnant Ewes. Conference Abstract, PAG-XXI, Jan 11-16. San Diego, CA. P614.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Chang, T.-C., Jiang, Y., Dalrymple, B., Archibald, A., Talbot, R., Kijas, J., Worley, K.C., McEwan, J.C., Cockett, N., Liu, W.-S. 2013. A contig map of the ovine Y chromosome. Conference Abstract, PAG-XXI, Jan 11-16. San Diego, CA. P619.
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Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: We have continued our research on the transcriptome of the bovine Y chromosome (BTAY) and identified a total of 18 known genes/families (12 single copy, and 6 multi-copy gene families) and 385 novel transcripts units (TUs). The majority of TUs are non-coding RNAs (ncRNAs). We conclude that the bovine Y chromosome male-specific region is gene-rich, which is contrary to previously held views that the Y chromosome is poor in gene content and limited in transcriptional potential. These BTAY-linked genes are expressed predominantly in testis and up-regulated during testis development and maturation. One of the BTAY gene families, PRAMEY, and its ortholog in the mouse, Pramel1, have been further characterized. We found both PRAMEY and Pramel1 mRNAs are expressed in testis only, and their proteins were mainly expressed in spermatids. Further analysis of the testis and spermatozoa indicated that the protein expression was coordinated with morphological alterations during acrosome formation in spermatids, and was different significantly in sperm tails in connecting piece, middle piece and principal piece. Our results suggest that PRAMEY and Pramel1 play a pivotal role in acrosome biogenesis and sperm motility. We have investigated the copy number variations (CNVs) of the PRAMEY gene family in 435 bulls from 15 cattle breeds, and carried out an association analysis of CNVs with bull reproductive traits. We applied quantitative real-time PCR to determine the copy number of PRAMEY using a single-copy Y-linked gene, DDX3Y, as the reference gene. The results revealed that the copy number of PRAMEY varied from 2 to 30 among all individuals analyzed with a significant difference among breeds (p<0.05). The median copy number of PRAMEY in taurine lineage (13 copies) is significantly lower than that of indicine lineage (17 copies). The lowest median copy number was observed in Holstein (12 copies), whereas the highest in Limous (24 copies). Association analysis with data from 261 Holstein A.I. bulls revealed that the CNVs were correlated negatively with the non-return rate (p<0.001), while positively with the semen quality index (p<0.05), suggesting that the CNV of PRAMEY may be a valuable genetic marker for bull fertility selection. A number of efforts also have been made to disseminate our research results and mentor graduate students. These include: i) presenting the results in national/international conferences such as SSR, ISAG and PAG; ii) sharing knowledge with cattle breeders and bull studs, especially with the leading companies, such as Select Sires and Genex, in the AI industry; iii) establishing collaborative researches with these companies with a purpose to evaluate the results and to transfer the knowledge into the cattle breeding and the A.I. industry; and iv) mentoring graduate students in the fields of ruminant reproduction and genomics. PARTICIPANTS: PSU: Wansheng Liu (PI), Ti-Cheng Chang (Post-doc), Yaqi Zhao (graduate student), Xiangpeng Yue (graduate student), Cooperating Investigators: Jon Oatley (Washington State University), Francisco Diaz (Penn State), and Hiroshi Yasue (National Institute of Agrobiological Sciences, Japan) TARGET AUDIENCES: Scientists in the fields of reproduction, male fertility, animal breeding and genetics, comparative genomics and molecular evolution. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Results from these studies provide details in: a) the gene repertoire of BTAY, which is fundamental for Y chromosome function and evolution study, b) the knowledge regarding the mRNA and protein expression of the bovine PRAMEY and the mouse Pramel1 during spermatogenesis, and c) CNVs of the bovine PRAMEY among cattle breeds. The data are essential for studying the functional role of PRAMEY/Pramel1 in spermatogenesis and male fertility, and shed a light on the possibility of using CNVs (of PRAMEY) for marker-assisted male fertility selection.
Publications
- Liu, W. S. 2012. The heterochromatic region of the bovine Y chromosome is gene-rich. PAG-XX. San Diego, CA. January 14-18, 2012. Conference Abstract. p. 634.
- Ponce de Leon, A. F. and W.-S. Liu. 2012. Bovine X and Y chromosomes. Chapter 7 in Bovine Genomics. Ed: James E. Womack, Wiley-Blackwell Publishing, USA. p.75-100.
- Mistry, B. V., T.-C. Chang, H. Yasue, M. Chiba, Y.-Q. Zhao, J. Oatley, and W.-S. Liu. 2012. Differential expression of PRAMEL1, a cancer/testis antigen, during spermiogenesis in the mouse. Molecular Reproduction and Development p. 1-6.
- Liu, W.-S., B. V. Mistry, T.-C. Chang, H. Yasue, M. Chiba, Y.-Q. Zhao, and J. Oatley. 2011. Differential Expression of PRAMEL1, a Cancer/Testis Antigen, during Spermiogenesis in the Mouse. The 45th Annual Meeting of the Society for the Study of Reproduction. SSR2012. State College, PA. August 12-15, 2012. Conference Abstract. p. 441.
- Chang, T.-C. and W.-S. Liu. 2012. The male-specific region of the bovine Y chromosome is enriched for testis-expressed non-coding RNAs. SSR State College, PA. August 12-15, 2012. Conference Abstract. p. 426.
- Yue, X.-P., T.-C. Chang, and W.-S. Liu. 2012. Copy number variations of PRAMEY are associated with bull fertility. SSR State College, PA. August 12-15, 2012. Conference Abstract. p. 462.
- Zhao, Y.-Q., B. V. Mistry, and W.-S. Liu. 2012. Expression analysis of the PRAME/PRAMEY gene family during bovine spermatogenesis. SSR2012. State College, PA. August 12-15, 2012. Conference Abstract. p. 428.
- Liu, W.-S., T.-C. Chang, and E. Retzel. 2012. Differential transcriptome of the bovine Y-chromosome is associated with testis development in cattle. 33rd International Society for Animal Genetics Conference. ISAG2012. Cairns, Australia. July 15-18, 2012. Conference Abstract. p. 2032.
- Liu, W.-S. and T.-C. Chang. 2012. Y-chromosome genomics: a comparison between the human, mouse, and bovine Y-chromosome. 3rd World DNA and Genome Day. WDD-2012. Xian, PR China. April 25-28, 2012. Conference Abstract. p. 50.
- Chang, T.-C., Y. Yang, E. Retzel, and W.-S. Liu. 2012. The genomic organization and gene content of the male-specific region (MSY) of the bovine Y-chromosome. PAG-XX. San Diego, CA. January 14-18, 2012. Conference Abstract. p. 573.
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