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Indian Journal of Biotechnology Vol 10, April 2011, pp 178-182 Exploration of Y-chromosome specific markers to discover SNP associated with sub fertility traits in dairy bulls C S Mukhopadhyay*, A K Gupta, B R Yadav and T K Mohanty Dairy Cattle Breeding Division, National Dairy Research Institute, Karnal 132 001, India Received 9 June 2009; revised 4 August 2010; accepted 29 October 2010 The present study was designed to identify single nucleotide polymorphism (SNP) in Y-chromosome specific genes, namely, sex determining region, Y-encoded (SRY) and testis specific protein, Y-encoded (TSPY) vis-à-vis, detection of association between the SNP and andrological parameters (like, conception rate and per cent individual motility of semen). A total of 106 bulls, belonging to Karan Fries (Holstein Friesian crossbreds cattle), Sahiwal cattle and Murrah buffalo, were selected based on poor reproductive performance (viz., libido, semen quality & freezability), along with some control bulls with normal andrological parameters. Single strand conformation polymorphism followed by sequencing of the different band patterns revealed only a single novel SNP (Guanine to Adenine transition) in the 4th intron of TSPY gene in Murrah buffalo. There was no significant association between the genotypes and the andrological parameters under study. Further study, encompassing larger number of animals distributed over many farms, could reveal sizeable number of SNP and association with spermatological parameters. Keywords: Buffalo, bulls Y-chromosome, SNP, sub fertility Introduction Bulls, the half of the herd, are selected at the age of 2-3 years for further reproduction and even a bull with appreciable production index is often culled due to some reproductive anomalies. The subfertility problems in dairy bulls, like inadequate libido and poor seminal profile, may be attributed to genetic, environmental and managemental causes. Genes responsible for sex-drive, spermatogonial proliferation and differentiation, sperm cytoskeleton structure, membrane structure and integrity, sperm viability, etc. are being identified and studied to shed light on the aforementioned problems. Scientists have screened half a dozen of holandric genes directly associated with fertility of the males1. Polymorphism study of these genes would help in the identification of some potential markers associated with the male fertility traits. Such studies have been conducted sporadically in bovines with sex-determining region, Y encoded (SRY) gene. Still, most of the directly associated genes (TSPY, RBMY, etc.) are yet to be —————— *Author for correspondence: Tel 91-161-2414023; Fax: 91-161-2400822 E-mail: [email protected] Present Address: Department of Animal Biotechnology, GADVASU, Ludhiana 141 004, India explored. The genomic DNA markers may be used to assess a bull’s reproductive efficacy at an early age, such as before the age of 6 months. The present investigation was designed to identify Y-chromosome specific markers associated with male reproductive traits. Single strand conformation polymorphism (SSCP) of two genes have been studied for the detection of polymorphism in cattle and buffalo bulls, namely, testis specific protein, Y encoded (TSPY) and sex-determining region, Y-encoded (SRY). Materials and Methods Selection of Animals A total of 106 animals were selected (Table 1) from the Artificial breeding complex (ABC), NDRI, Karnal, on the basis of their reproductive performance. The subfertile bulls were categorized as poor libido (not showing adequate sex drive for at least three consecutive months), poor semen quality (individual motility < 40% and watery appearance of the neat semen) and poor semen freezability (with post thaw motility < 35% following one d of cryopreservation). Some control bulls (with normal libido and acceptable semen quality) under each breed were taken. Peripheral blood and semen samples (from semen donating bulls) were collected from these animals. MUKHOPADHYAY et al: Y-CHROMOSOME SPECIFIC SNP 179 Table 1—Per cent individual motility and number of bulls (data in parenthesis) under different species-breeds vis-à-vis fertility groups taken in the study Reproductive performance Poor libido Poor semen quality Poor semen freezability Control Total Total samples Karan Fries Sahiwal Murrah (8) 20.000 ± 2.289 (23) 18.750 ± 3.504 (10) 68.696 ± 0.718 (23) (64) (4) 37.500 ± 2.500 (4) 37.500 ± 2.500 (4) 68.333 ± 1.666 (6) (18) (106) (6) 22.500 ± 4.7871 (4) 40.000 ± 0.000 (4) 65.556 ± 1.7568 (10) (24) Collection of Semen Semen samples were collected from each bull at weekly intervals. The per cent individual motility of each sample was recorded immediately after collection (Table 1). The available per cent conception rates (CR%) of the bulls were also recorded from the artificial insemination register. DNA Extraction Genomic DNA was isolated from blood using standardized protocol of Phenol-chloroform extraction technique2. Quality and quantity of the extracted DNA was checked by horizontal submarine mini electrophoresis (Biorad, USA) in 0.8% agarose (Sigma, USA), using 0.5X TBE as running buffer and measuring the optical density (OD) at 260 nm and 280 nm wavelengths in UV-VIS spectrophotometer (Hitachi, Model U-1500, Japan) against autoclaved HPLC water as blank sample. Primers and Optimization of PCR Primer3 online software3 was used to design the primers of TSPY gene taking Bos taurus sequence as template. Primers for SRY gene were taken from Kato et al4. Same set of primers (Table 2) (Genetix Inc. New Delhi) were used for PCR amplification of both cattle and buffalo templates. PCR amplification of the template DNA was carried out in thermal cycler (Biometra, Germany), programmed accordingly for each primer pairs. The composition of the master-mix was as follows: 60 ng of template DNA, 0.8 units of Taq polymerase (Bangalore Genei), 0.2 mM of dNTPs and 0.5 µmol primers (forward and reverse, each), 1X PCR buffer with 1.5 mmol MgCl2. Single Strand Conformation Polymorphism (SSCP) Five µL of amplicon was mixed with 8 µL of loading dye, denatured at 95°C for 5 min and electrophoresed on a SSCP gel in vertical electrophoretic unit (Bio-rad Protean II xi Cell, USA). Different concentrations of non-denaturing gel (10, 12 & 14%) were used depending on the size of the amplicon. Gel was run at a constant voltage (200 V) for 8-12 h (depending on amplicon size) at 4°C. The gel was fixed in 10% glacial acetic acid for 30 min, stained with freshly prepared 1% silver nitrate solution, at dark for 40 min. Gel was immediately transferred to freshly prepared developing solution (3% sodium carbonate, 10 mL formaldehyde, 0.001% sodium isothiocyanate) following a brief washing in glass distilled water. Various band patterns of the amplified PCR products were marked and scored. Sequencing of SSCP Variants and Establishment of Gene Identity The variants were further cross-checked by repeating the SSCP and the gene variants were sent for sequencing to Bangalore Genei (Bangalore, India). Sequence of the variants obtained was edited using Bio Edit Version 7.0.9.05. ClustalW (1.83) multiple sequence alignment software6 was used to annotate the consensus sequences for different band patterns to find out single nucleotide polymorphism (SNP). Statistical Analysis Alignment study of the sequences of the SRY and TSPY gene was done using MegAlign software (DNASTAR, USA). The phylogenetic trees were constructed following unweighted pair group method for arithmetic mean (UPGMA) for 1000 bootstrap values7, using CLC-Free Workbench Software. The data generated on various andrological parameters (libido, CR% and per cent individual motility) were subjected to least squares analysis to detect any significant difference among the SSCP variants (band patterns) for the parameters under study. The CR% and per cent individual motility data were arc-sine transformed before analysis8. Systat Version 6.0.1, 1996, SPSS Inc was used for factorial analysis. The statistical model used was: Yij = µ + Bi + eij When, Yij = andrological parameter (libido or arcsine CR% or arc-sine individual motility) of jth semen sample showing ith band pattern; µ = overall INDIAN J BIOTECHNOL, APRIL 2011 180 population mean; Bi = Effect of ith band pattern (i= 1, 2, 3); eij= residual error. Results and Discussion Variations in the SSCP band patterns were observed for all the primers, except for SRY 3’-UTR, TSPY-12 and TSPY-23 for those genetic groups (Table 3). The nucleotide sequences of different fragments of the Y-chromosome specific genes obtained were submitted to NCBI Genbank (Table 3). A single SNP was discovered in Murrah in the TSPY 45 fragment (fourth intronic region) due to transition of Guanine to Adenine at nucleotide 211 and 210 of Variant I and III, respectively (Fig. 1). Types I and II variants were virtually not different. Variant I was found to be more prevalent (92% of the total sample). Hence, Variant III was considered to be a mutant type. The nucleotide sequence of Murrah was subjected to alignment study which revealed maximum homology with B. taurus sequence (90.3%) (Fig. 2). Factorial analysis did not reveal any significant difference among the variants for any of the andrological traits. The animals selected for the present study were taken from a single herd on the basis of their seminal Table 2—Specifications of the primers used for PCR-SSCP study Primer Name Primer Sequence TSPY-12F TSPY-12R TSPY-23F TSPY-23R TSPY-34F TSPY-34R TSPY-45F TSPY-45R TSPY-55F TSPY-55R TSPY-66F TSPY-66R SRY-HMG -F 5’-TAGATGCCCTGCAGGCACTGG-3’ 5-TTCAAGTCCATCATGGTAGCCG-3’ 5’-TCAAGTTTCCATCCTGATCAG-3’ 5’-ATGTCAAGGTAATACTCCTTA-3’ 5’-TCCGCCGTCCCGCTGCAAGCT-3’ 5’-CAATCCTGTTCGATTCTGGGC-3’ 5’-GTCGATCCACCCCAGTCCACT-3’ 5’-ACCCCATCACCACATGTGTTT-3’ 5’-GTCCATGAGAGACAACTGAC-3’ 5’-GTCGTATCACCTAGGACTCTC-3’ 5’-ACCCCTGTGTTCCCTGTTT-3’ 5’-ATACGGTCATGCGGAGAGT-3’ 5’-CCATGAACGCCTTCATTGTG-3’ SRY-HMG-R 5’-GCTCTCCGACGAGGTCGATA-3’ 58.2 SRY3’UTR-F SRY3’UTR-R 5’-TGGTCCTCTGTTAATTAGTTCT-3’ 5’-TGGATGTTATTAATCGCCTC-3’ 50.8 49.7 Tm(°C) 65.0 62.0 57.0 54.0 69.0 61.0 65.0 59.0 58.0 61.0 57.0 57.0 54.6 Target (bp) Reference 255 XM_871389 176 EF432553 236 EF432553 236 U75896 290 AY347587 230 AY347587 217 Kato et al 1995 513 Table 3—Different SSCP variants (per cent) recorded in SRY and TSPY genes S.No. Breeds Primer NCBI Accession No. Percent each variant 1. Sahiwal SRY-HMG EU386186 2. 3. 4. 5. K.F. Murrah Sahiwal K.F. Murrah Sahiwal K.F. Murrah Sahiwal K.F. Murrah Sahiwal K.F. Murrah TSPY 34 TSPY 45 TSPY 55 TSPY 66 ------------EU350952 EU032586 --------EU386187 EU386-----188 ----EU326528 ----EU386185 ----EU386184 Type I (55.56%) Type II (44.44%) Monomorphic Type I (80.00%); Type II (12.00%); Type III (8.00%) Type I (77.78%); Type II (22.22%) Type I (82.81%); Type II (12.50%); Type III (4.69%) Type I (72.00%); Type II (4.00%); Type III (24.00%) Type I (83.33%); Type II (16.67%); Monomorphic Type I (52.00%); Type II (40.00%); Type III (8.00%) Monomorphic Type I (39.06%); Type II (53.13%); Type III (7.80%) Type I (84.00%); Type II (8.00%); Type III (8.00%) Type I (88.89%); Type II (11.11%) Monomorphic Type I (84.00%); Type II (16.00%) Values in parentheses indicate the frequency percentage of individual variant MUKHOPADHYAY et al: Y-CHROMOSOME SPECIFIC SNP 181 Fig. 1—SSCP band patterns of TSPY 45 fragment in Murrah buffalos. Fig. 2—Alignment of TSPY gene (Exon 4- Exon 5) with other species and the phylogenetic tree (Nucleotide alignment) (1000 bootstraps) by UPGMA. performance. The experimental animals do not represent a random sample, nor do they reflect the overall genetic make-up of the population maintained at NDRI herd, since the males were selected as future bulls through a multi-stage selection procedure. More than 80% of the male calves born are auctioned within six months based on their expected predicted difference (EPD) and growth rate. Results in both K F and Sahiwal did not reveal any polymorphism at the nucleotide level for any of the gene fragments. The SNP detected at 4th intron of Murrah buffalo was novel, though it was not found to be associated with spermatozoal parameters. However, the results does not dictate to consider that SRY or TSPY are not associated with spermatological profile of bulls. SRY gene has been well studied in human, mice and cattle. A number of reports on the polymorphism of SRY gene in humans suggested that point mutations in the SRY-HMG result in sex-reversal and gonadal hypoplasia of the males9,10. No report is available on the association of SRY gene with libido of animals or human beings. In a polymorphism study to find out SSCP variants of SRY gene, the result showed that mutation is associated with oligospermia in man11. Jayakumar12 studied polymorphism of 48 Murrah buffalo bulls, maintained at NDRI herd and adjoined field samples, and reported nine variants. The report on association of TSPY with semen freezability is meager. However, the selected spermatological traits (% individual motility) and conception rates (CR %) were hypothesized to be associated with this gene, since the gene is associated with spermatogonial proliferation. A few reports have explored the existing polymorphism of TSPY in bovines. Verkaar et al13 reported sequence variation within multicopy gene family at 4 different points of intron 5 (AY347587) of B. taurus and at 2 points of intron 5 of Bos taurus (AY347589). In the present study, out of 24 Murrah bulls studied, 2 bulls (Mu-5083 and Mu-5525) found as type III genotype, were the mutant type. The association study between the genotypic variants in Murrah with per cent individual motility as well as conception rates (CR %) was not found significant. Performance records of those two bulls revealed varying reproductive profile. Mu-5083 yielded freezable ejaculates with normal reproductive performance. While Mu-5525 buffalo bull used to show poor libido and had some off-bred characteristics. It was disposed due to stunted growth. Similar type of work has not been accounted in other species, though it has been reported that TSPY is functionally coherent to spermatogenesis and fertility14-16. TSPY being expressed in spermatogonia and to a lesser extent in primary spermatocytes17 has a role in spermatogonial proliferation. The multiple sequence alignment study was done to find out the extent of homology of the nucleotide sequence of TSPY- 45 fragment in Murrah with those INDIAN J BIOTECHNOL, APRIL 2011 182 of other closely related species. The present study demonstrated at least 80% conservation among the bovidae species. Comparative mapping among several species including human, mouse, cattle, sheep, dog, lemur and Sminthopsis macroura has shown that the Y chromosome PAR genes are not conserved across mammalian species18. bTSPY is located in the region of the Y chromosome that does not take part in recombination. TSPY gene families of cattle, sheep and goat18,19 are much more closely related to each other than to TSPY of other artiodactyls14. Although no apparent similarity in the banding pattern exists with regard to the sex-chromosomes of cattle, sheep and goat21,22, gene composition seems to be largely conserved. Thus, genetic homology of the Y chromosomes is maintained over this evolutionary distance in spite of extensive structural diversification. Conclusion The single nucleotide polymorphism identified in the 4th intron in Murrah buffalo is a novel SNP, yet it did not reveal any significant association with CR % and per cent individual motility of the semen. SRY and TSPY homologues have been shown to exist in several other mammalian species. Further study is required to identify Y-chromosome specific markers associated with andrological parameters in farm animals. Acknowledgement The authors are thankful to the Director, NDRI, for providing financial assistance for the research work. 5 6 7 8 9 10 11 12 13 14 15 16 17 18 References 1 2 3 4 Xiao C, Tsuchiya K & Sutou S, Cloning and mapping of bovine ZFX gene to the long arm of the X-Chr (Xq34) and homologous mapping of ZFY gene to the distal region of the short arm of the bovine (Yp13) ovine (Yp12-p13) and caprine (Yp12-p13) Y chromosome, Mamm Genome, 9 (1998) 125-130. Shashikanth, A study on DNA polymorphism in cattle and buffaloes. Ph D Thesis, National Dairy Research Institute, Karnal, India, 1999. http//frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_slow.cgi Kato Y, Sato S, Cui X, Itagaki Y & Sutou S, Cloning and characterization of bovine Sry gene, Anim Sci Technol, 66 (1995) 994-1001. 19 20 21 22 Hall T A, BioEdit a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT, Nucleic Acids Symp Ser, 41 (1999) 95-98. http//www.ebi.ac.uk/Tools/clustalw /index.html Sneath P H A & Sokal R R, Numerical taxonomy, Nature (Lond), 193 (1962) 855-860. Snedecor G W & Cochran W G, Statistical methods. 8th edn (Iowa State University Press, Ames, Iowa) 1989, 1-289. Ferrari S, Harley V R, Pontiggia A, Good Fellow P N, LovellBadge R & Bianchi M E, The SRY like HMG1 recognizes sharp angles in DNA, Embo J, 11 (1992) 4497-4506. King C Y & Weiss M A, The SRY high mobility group box recognizes DNA by partial intercalation in the minor groove A topological mechanism of sequence specificity, Proc Natl Acad Sci USA, 15 (1993) 11990-11994. Ozdemir O, Gull E, Kilicarslan H, Gokce G, Beyaztas F Y et al, SRY and AZF gene variation in male infertility: A cytogenetic and molecular approach, Intl Urol Nephrol, 39 (2007) 1183-1189. Jayakumar S, Molecular characterization of SRY gene in Murrah buffaloes, MVSc Thesis. National Dairy Research Institute, Karnal, India, 2006. Verkaar E L, Zijlstra C, van't Veld E M, Boutaga K, van Boxtel D C & Lenstra J A, Organization and concerted evolution of the ampliconic Y-chromosomal TSPY genes from cattle, Genomics, 84 (2004) 468-474. Vogel T, Borgmann S, Dechend F, Hecht W & Schmidtke J, Conserved Y-chromosomal location of TSPY in Bovidae, Chromosome Res, 5 (1997) 182-185. Vogel T, Dechend F, Manz E, Jung C, Jakubiczka S et al, Organization and expression of bovine TSPY, Mamm Genome, 8 (1997) 491-496. Vogel T, Dittrich O, Mehraein Y, Dechend F, Schnieders F & Schmidtke J, Murine and human TSPY genes novel members of the TSPY-SET-NAP1L1 family, Cytogenet Cell Genet, 81 (1998) 265-270. Schnieders F, Dork T, Arnemann J, Vogel T, Werner M & Schmidke J, Testis-specific protein Y-encoded (TSPY) expression in testicular tissues, Hum Mol Genet, 5 (1996) 1801-1807. Graves J A M, Wakefield J & Toder R, The origin and evolution of the pseudoautosomal regions of human sex chromosomes, Hum Mol Genet, 7 (1998) 1991-1996. Jakubiczka S, Schnieders F & Schmidtke J, A bovine homologue of the human TSPY gene, Genomics, 17 (1993) 732-735. [Erratum published in Genomics (1994) 191 198]. Schempp W, Binkele A, Arnemann J, Glaser B, Ma K et al, Comparative mapping of YRRM- and TSPY-related cosmids in man & hominoid apes, Chromosome Res, 3 (1995) 227-234. Di Berardino D, Hayes H, Fries R & Long S, International system for cytogenetic nomenclature of domestic animals, Cytogenet Cell Genet, 53 (1989) 65-79. Hediger R, Ansari H A & Stranzinger G F, Chromosome banding and gene localizations support extensive conservation of chromosome structure between cattle and sheep, Cytogenet Cell Genet, 57 (1991) 127-134.