Document Type : Original Article
1 Department of Animal Science, Agricultural Faculty, Khorasgan Branch, Islamic Azad University, Isfahan, Iran
2 Biology Department, School of Sciences, University of Isfahan, Isfahan, Iran;Departments of Cell and Molecular Biology, Cell Science Research Center, Royan Institute for Animal Biotechnology, ACECR, Isfahan, Iran
3 4Young Researchers Club, Khorasgan Branch, Islamic Azad University, Isfahan, Iran.
4 5MRC Centre for Reproductive Health Queen's Medical Research Institute, University of Edinburgh, Scotland, UK
Normal expression of the oocyte specific genes
growth differentiation factor-9 (GDF9), located
on chromosome 5 and bone morphogenetic protein
(BMP15), also known as GDF-9B , located on
the X chromosome (
Even though the mutation in autosomal BMPR1B additively increases sheep fecundity, some
GDF9 mutations enhance ovulation rates only in
heterozygous animals (
There are high variations among different Iranian
sheep breeds in terms of carcass yield and prolificacy.
Twin births are frequent in some breeds
though infertility is rarely observed in these flocks.
Iranian sheep flocks have been analysed for mutations
in these major fecundity genes over the last
decade but no significant mutations were detected
This study was performed on Iranian Afshari sheep screened GDF9 mRNA extracted from slaughtered ewe ovaries classified in terms of the degree of follicle development on external morphological appearance, and reports the presence of 3 previously known GDF9 mutations, one of which is associated with increased fertility.
All the following procedures which were carried out on animals were approved by the Animal Welfare Committee and the Halal Commission of Khorasgan Branch, Islamic Azad University.
Since we did not have a reliable database of sheep
fecundity trait, follicular and morphological status
of ewe ovaries were considered as an indicator
for ovulation rate and its consequential litter size.
After slaughtering 30 ewes, ovaries were placed
in normal saline and transferred to the laboratory
within 2-3 hours where they 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 the 30 pairs of ovaries, 10 showed
excellent and another 10 showed poor follicle
numbers and thus were assigned to excellent and
poor groups respectively. Homozygote and heterozygote
genotypes for either BMP15 or GDF9
have been considered to result in sterility and high
fecundity, respectively (
Eight ovaries each from poor, good, and excellent groups were selected at random for histological evaluation. Following fixation in 10% PFA, ovaries were sectioned into 5 microns using microtome and underwent hematoxylin and eosin staining procedure to discriminate nucleus and cytoplasm. Slides were deparaffinized and rehydrated in descending graded series of alcohol and distilled water. Following hematoxylin staining, destaining was performed in acid-ethanol and distilled water. Finally, slides were stained with eosin and dehydrated in graded ethanol concentrations.
Thin slices of ovaries were immediately thawed using the freeze-thawing process followed by RNA extraction in 1 ml of AccuZol (#K3090, Bioneer) and 100 μl of chloroform. The mixture was centrifuged for 15 minutes 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 minutes and dissolved in 25 μl of DEPC treated water. Presence of a unique RNA pattern on agarose gel electrophoresis indicated a high quality of extracted RNA.
Reverse transcription (RT) step was conducted
using RevertAid First St cDNA kit (#EP0441,
Fermentas) with minor modifications. Briefly, 1
μl (3 μg) 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 minutes,
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 minutes, 42°C for 60 minutes, and 70°C for 5
minutes. One micro litre of the cDNA was used
for PCR reaction to amplify a 589 bp fragment
and (5'-CAATTCAGAGCTGGCACTCTCC-3') as forward and reverse primers, respectively (
Sections at different parts of each ovary from poor,
fair, and excellent ovaries were used for histology.
Though no follicles were detected on the surface of
poor ovaries, the histology examination revealed
the presence of follicles at all different stages of
growth and development and there was no indication
of any failure of follicle development as seen in
e.g. homozygous Thoka GDF9 mutant sheep (
To determine the potential expression of GDF9
mutations among different initial classifications of
ovaries, RNA extraction and reverse transcription
procedures for GDF9 gene were conducted (
GDF9 PCR from sheep ovaries genome (DNA) and cDNA (A), and sequencing results for GDF9 mRNA showed G2, G3, and G4 mutations (B).
Amino acid alignment for G4 (G721A) mutation which leads to nonconservative E241K shift in unprocessed amino acid peptide.
Sequencing results from the GDF9 cDNA showed
that there were 3 point mutations compared to
NM_001142888 code in 1 out of 12 ovaries (
Abundance of G2, G3, and G4 SNPs in 12 sheep mRNA
|Embryo quality||Sample size||G2 (C471T)||G3 (G477A)||G4 (G721A)|
Lack of registered records for fertility traits in
Iranian sheep flocks has been considered as the
main obstacle for major gene detection. Our investigation
of different flocks for sterile ewes
indicated that repeated signs of oestrous (heat),
without pregnancy is rare. However the lack of reliable
breeding records resulting in uncertainty in
determining high prolificacy ewes forced us to use
follicle number as potential indirect sign for ovulation
rate and litter size. Histological assessment
of more than 10 poor ovaries indicated that there
were numerous follicles at all stages of development
inside white ovaries which makes them comparable
in terms of follicle development to high
quality ovaries. This indication from abattoir-derived
ovaries plus the very low incidence of infertility
suggests that significant mutations in GDF9
which led to infertility and high prolificacy in
Belclare and Cambridge (
In accordance to Hanrahan et al. (
Other investigations of Iranian breeds showed
that major mutations on twining rate are not the
case for higher prolificacy of Iranian sheep (
So far, presence of the G1 (
Twin births in some Iranian sheep breeds are
common, though 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 the G4 mutation was not supposed
to significantly affect prolificacy in sheep flocks
having more important mutations (