Download growth differentiation factor 9 (GDF9) gene is located t the 5 th

Title:
Presence of SNPs in GDF9 mRNA of Iranian sheep
Authors: Shahin Eghbalsaied1*, Somayeh Shahmoradi2, Akbar Pirestani1,
Kamran Ghaedi3,4, and Hamidreza Amini2
1
Department of Animal Science, Agricultural Faculty, Khorasgan branch,
Islamic Azad University, Isfahan, Iran.
2
Young Researchers Club, Khorasgan branch, Islamic Azad University, Isfahan,
Iran.
3
Department of Genetic, Reproductive Medicine Research Centre and Cell science
research centre, Royan Institute (Isfahan Campus), ACECR, Tehran, Iran
4
Biology Department, School of Sciences, University of Isfahan, Isfahan, Iran
Correspondence: Shahin Eghbalsaied, Department of Animal Science,
Agricultural Faculty, Khorasgan branch, Islamic Azad University, Isfahan, Iran.
P. O. Box: 8166117677
Correspondence E-mail address: [email protected]
Telephone No: +98-9131676736
Fax No: +98-311-5354015
Abstract
Twining birth is almost prevalent in some Iranian sheep breeds, while infertility
scarcely occurs. Mutation detection in major fecundity genes has been explored in
most of Iranian sheep flocks since last decade. However, predefined efficient mutant
alleles for BMPR-1B and GDF9 have not been detected. This study was aimed to
screen GDF9 mRNA extracted from slaughtered ewe ovaries among different sources
of Iranian sheep breeds. Ovaries with poor, fair, and excellent quality were used for
histology and RNA extraction processes. High quality RNAs underwent reverse
transcription. No streak ovary which has been considered as infertility sign due to
mutant homozygous for GDF9 and BMP15 was observed. Sequencing results from
the GDF9 cDNA showed that G2 (C471T), G3 (G477A), and G4 (G721A) mutations
were observed from 1, 4, and 1 out of 12 ewes, respectively. Though all 3 mutations
were previously reported, this is the first report on their presence in Iranian breeds.
The first and second mutations are at the third codon bases and subsequently
inefficient though E241K occurred because of G4 SNP. As the G4 mutation was
observed in an excellent ovary, it might induce ovulation rate in Iranian sheep.
Moreover, aggregative effect of minor mutations in GDF9 and BMP15 mRNAs might
be the reason for relatively higher fecundity in some Iranian sheep breeds.
Keywords: GDF9, SNP, sheep, Iran
Introduction
Growth differentiation factor-9 (GDF9) or bone morphogenetic protein-9B (BMP-9B)
gene is located on chromosome 5 of sheep. It includes two exons in 1126 bp length
and encodes a premature peptide with 453 amino acids which in its mature form
contains 135 amino acids (Sadighi et al., 2002). Proper expressions of both GDF9 and
BMP15 in sheep ovaries are necessary for follicllogenesis fulfilment and their
inactivation lead to a great halt in follicle development at type 2 stage (Juengel et al.,
2002; Nicol et al., 2009). GDF9 level can affect on the corpus leuteum function and
subsequently follicular development and pregnancy (Juengel et al., 2000). Moreover,
the bone morphogenetic protein receptor-1B (BMPR-1B) mutation induces
precocious maturation of ovarian follicles by increasing the sensitivity of the follicles
to FSH without increase in FSH concentrations (Bogarti, 2009).
Even though mutation in autosomal BMPR-1B additively increases sheep fecundity,
GDF9 mutations enhance ovulation rate in over dominance manner (Hanrahan et al.,
2004). However, recent publication indicates the presence of fertility in mutant
homozygous for either GDF9 or BMP15 genes (Barzegari et al., 2010). So far, eight
point mutations in Belclare and Cambridge breeds (Hanrahan et al., 2004), and one
more mutation in Thoka breed (Nicol et al., 2009) were reported for GDF9 gene.
There are high variations among different Iranian sheep breeds from carcass yield and
prolificacy aspect. Twining rate is almost prevalent in some breeds though the
infertility is hardly observed in the flocks. Iranian sheep flocks have been subjected
for mutation detection in major fecundity genes since last decade (see DeldarTajangookeh al., 2008, for review). However, predefined mutant alleles with neither
additive inheritance (BMPR-1B) nor over-dominance manner (BMP15 and GDF9)
were detected in Iranian Shaal and Ghezel breeds, respectively (Deldar-Tajangookeh
al., 2008; Akbarpour et al., 2008). However, recent report indicates the presence of
G1 and B2 mutations in GDF9 and BMP15 genes respectively, in Moghani and
Ghezel breed (Barzegari et al., 2010).
The present study was aimed to screen GDF9 mRNA extracted from slaughtered ewe
ovaries among different sources of Iranian sheep breeds.
2. Materials and methods
2. 1. Samples
Since we did not have a reliable database of sheep fecundity trait, follicular and
morphological status of ewe ovaries were considered as clues for ovulation rate and
its consequential litter size. Collected ovaries were transferred in normal saline to the
lab, 2-3 h after slaughtering. Ovaries were classified into 3 categories based on follicle
number including poor (no observable follicles on the surface), good (regular), and
excellent (containing abundant follicles). Among those 30 pairs collected ovaries, 10
and 10 were belonged to excellent and bad groups.
2. 2. Histology
Eight ovaries from poor, good, and excellent groups were selected. Three sections
from right and left ovaries were randomly cut and used for histology. Following
fixation in 10% PFA, 5 micron sections diameter were excised and used for histology.
Briefly, the ovarian tissues were fixed in 4% (w/v) paraformaldehyde in PBS (pH
7.2), processed for histology, and embedded in paraffin wax. Paraffin wax was
removed from the 5 pm sections using xylene and they were rehydrated in a graded
series of ethanol. Endogenous peroxidase was blocked by incubating the sections in
3% (v/v) hydrogen peroxide in methanol.
2. 3. RT-PCR
Thin slices of ovaries were immediately underwent freeze-thawing process followed
by RNA extraction in 1 ml of AccuZol (#K3090, Bioneer) and 100 μl of chloroform.
The mixture was centrifuged at 13,500 rpm for 15 min at 4˚C. Equal volume of
Isopropyl alcohol in addition to 1μl glycogen (RNA grade, #0551, Fermentas) were
added to the supernatants and stored at -20˚C for 2 hours. After removing of Isopropyl
alcohol, washing steps by ethanol were repeated. The RNA pellet was air dried at
37˚C for 5 min and dissolved in 25 μl of DEPC treated water. Presence of a unique
RNA pattern on agarose gel electrophoresis indicated as high quality of extracted
RNA.
Reverse transcription (RT) step was conducted using RevertAid First St cDNA kit
(#EP0441, Fermentas). Briefly, 1 μl of total RNA and 1 μl of random hexamer
primers were added into 10 μl of DEPC water. The mixture was incubated at 70 ˚C for
5 min, chilled on ice, and mixed with RT ingredients including 5X reaction buffer,
dNTP mix (200 μM), RNase inhibitor, and RT enzyme (1 μl). The cDNA was
synthesized via incubation at 25 ˚C for 5 min , 42 ˚C for 60 min, and 70 ˚C for 5 min.
One micro litre of the cDNA was used for PCR reaction to amplify a 589 bp fragment
with
(5’-CAACACTGTTCGGCTCTTCACC-3’)
and
(5’CAATTCAGAGCTGGCACTCTCC-3’) as forward and reverse primers, respectively
(McAlen et al., 2010). PCR reaction mixture in total 25 μl contained: 50 ng of cDNA
dissolved in DEPC-treated water, forward and reverse primers (10 pM), dNTPs
mixture (200 μM each), 10X PCR buffer, 50 mM magnesium chloride, and 0.5 unit of
Ex-Taq DNA Polymerase (#RR01, TaKaRa). PCR cycling conditions initiated at
94˚C for 4 min, followed by 35 cycles of 94˚C for 30 s, 59˚C for 20 s, and 72˚C 30 s,
and ended by an extra extension at 72˚C for 7 min. The resulting PCR products were
electrophoresed on 1% agarose gel which was pre-stained with ethidium bromide (0.5
μg/ml), and photographed using UVITECH transilluminator. PCR products from 12
ewe ovaries which showed favourite band were sent for sequencing.
3. Results
3. 1. Histology
Sections at different parts of each ovary from poor, fair, and excellent ovaries were
used for histology part. Though no follicle was detected on the surface of poor
ovaries, the histology examination revealed presence of follicles at different growth
steps. Our seeking to find poor ovary without high stage follicles which has been
indicated as sterility was not successful.
3. 2. RT-PCR and sequencing
To fulfil our knowledge on functional variation among different ovaries RNA
extraction and reverse transcription procedures for GDF9 gene were conducted
(Figure 1). Sequencing results from the GDF9 cDNA part showed that there were 3
point mutations compared to NM_001142888 code in 1 out of 12 ovaries (figure 1).
This is the first report of presence of G2, G3, and G4 mutations in Iranian sheep
breeds. Detected sequences from forward and reverse primers were different in these
3 bases which indicated as heterozygousity and confirmed by repeated sequencing.
The first and second mutations were C471T and G477A which both are at the third
base of codons and subsequently could not change the amino acid polypeptide
sequence. However, there was a significant shift in the amino acid sequence (E241K)
due to swapping of G at base 471 with A which cause non-conservative amino acid
change (figure 2). Frequencies of G2, G3, and G4 SNPs in 12 sequenced amplicons
were 1, 4, and 1 case, respectively (table 1). Interestingly only a high quality ovary
contained all three mutations. All sheep were heterozygous for detected mutations and
no homozygous case was detected.
4. Discussion
Lack of registered records for fertility traits in Iranian sheep flocks has been
considered as the main obstacle for major gene detection. Our investigation in
different flocks for sterile ewes indicated that there is rare event of repeated heat signs
without pregnancy. This uncertainty in determining high prolificacy ewes enforced us
to use follicle number as reliable indirect sign for ovulation rate and litter size.
Histological assessment of more than 10 poor ovaries indicated that there were
numerous numbers of follicles inside of white ovaries which make them comparable
to high quality ovaries. This indication from abattoir derived ovaries plus ignorable
infertile events pointed out that significant mutations which led to infertility and high
prolificacy in Belclare and Cambridge breeds (Hanrahan et al. 2004) might not be the
case for twining rate in Iranian breeds.
In accordance to Hanrahan et al. (2004), our results showed that there are three
mutations in Iranian breeds. Both of G2 (C471T) and G3 (G477A) mutations caused
non conservative substitutions in translated amino acid. However, G4 mutation
replaces glutamic acid to lysine at amino acid residue 241 of the unprocessed protein,
and leaves a basic group in place of an acidic group. Though this SNP occurs at a
position before the furin processing site and was supposed to have ignorable effect on
fertility (Hanrahan et al., 2004), its occurrence in high quality ovaries could be
effective for fecundity in Iranian breeds. The effect of G1 mutation was
underestimated previously (Hanrahan et al., 2004), while recent documents showed
that homozygote ewes for G1 mutation in Iranian Ghezel breed caused infertility
(Barzegari et al., 2010). Other investigation on Iranian breeds showed that major
mutations on twining rate are not the case for higher prolificacy of Iranian sheep (see
Deldar-Tajan Gookeh et al., 2008, for review). In summary, none of GDF9, BMP15,
and BMPR-1B detected mutations was observed in Shal breed which had the highest
litter size among Iranian sheep breeds. Neither BMP15 nor BMPR-1B muations was
the high prolificacy of Iranian Lori-Bakhtyari and Ghezel breeds. Moreover, G8
mutation which caused over dominance phenotype in Belclare and Cambridge breeds
was not detected in Iranian breeds (Akbarpour et al., 2010).
So far presence of SNPs in G1 (Barzageri et al., 2010) and G4 (present study) have
been reported to be effective for Iranian sheep prolificacy. This might be due to
change in GDF9 propeptide in which could act as GDF9 inhibitor (Jiang, et al., BBRC
315:525-531 (2004); Gregory et al., J. Biol. Chem. 280:27970-27980 (2005).
Moreover, ssummation of mutations with minor effects in the whole mRNA sequence,
even in unprocessed peptides, could induce twining rate in Iranian breeds.
Conclusion
Twining rate in some Iranian sheep breeds is common, though the infertility is
scarcely detected. Our investigation on GDF9 mRNA extracted from abattoir derived
ovaries showed that there were 3 point mutations including two conservative
substitutions, G2 (C471T) and G3 (G477A), and one non-conservative mutation, G4
which replaces glutamic acid to lysine (E241K) in the unprocessed protein. Though
G4 mutation was not supposed to be significantly effective on sheep prolificacy in
sheep flocks having more important mutations (Hanrahan et al., 2004), its presence in
high quality ovaries in Iranian sheep might indicate its importance for twining rate.
Moreover, aggregative effect of minor mutations in the whole mRNA sequence
including both unprocessed and processed peptides might be the reason for relatively
higher fecundity in some Iranian sheep breeds. However, screening of major genes
including GDF9, BMP15, and BMPR-1B in their whole parts like un-translated
regions in all Iranian breeds are needed for confirmation.
Acknowledgment
This project was funded by Islamic Azad University, Khorasgan branch, Isfahan, Iran.
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Table 1. Abundance of G2, G3, and G4 SNPs in 12 sheep mRNA
Embryo quality Sample G2 (C471T) G3 (G477A) G4 (G721A)
size
Poor
3
0
3
0
Fair
5
0
0
0
Excellent
4
1
1
1