Download IJBT 10(2) 178-182

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.
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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.