Genetic polymorphism of growth differentiation factor 9 (GDF9) gene related to fecundity in two Egyptian sheep breeds

Abstract

Purpose

This study explores polymorphisms in the growth differentiation factor 9 (GDF9) gene (exon 1) with respect to fertility in Egyptian sheep.

Methods

Blood samples were collected, and genomic DNA was extracted from 24 Saidi and 13 Ossimi ewes. A 710 bp portion of the GDF9 gene, was amplified using specific primers, and the sequence was analyzed to clarify the phylogenetic relationship of Egyptian breed sheep. In addition, the PCR-RFLP method using Pst1 or Msp1 restriction enzymes was used to mask polymorphisms of partial exon 1 of GDF9 gene to establish molecular markers for twinning.

Results

The lambing rate percentage and litter size showed significant difference between ewes, which produce single and twin lamb for each breed individually, whereas the coefficient of variation of the Saidi breed is greater than that of the Ossimi breed. The results suggested that the GDF9 gene shared a similarity in sequence compared to six accession numbers of Ovis aries found in GenBank. Molecular phylogenetic analyses were performed based on nucleotide sequences in order to examine the position of the Egyptian breeds among many other sheep breeds. The results indicate that accession number AF078545 of O. aries is closely related with Saidi and Ossimi ewes that produce single or twin lamb using the unweighted pair group method with arithmetic mean (UPGMA) analysis. Results showed that Msp1 enzyme digestion revealed polymorphic restriction pattern consisting of one band with 710 bp for ewes producing single lamb and two bands with 710 and 600 bp for ewes producing twin lamb in Saidi sheep breed.

Conclusion

Sequence analysis and diversity of polymorphisms in the GDF9 gene (exon 1) have a novel base substitution (A–T) for detection of Fec^G mutations that serve as a molecular marker for twinning.

Introduction

The main objective of sheep breed in the world is one or more of the following: meat, milk, and wool production, where in Egypt, the sheep meat production is more important than fiber production and the sheep contribute 6% of the total red meet produced [1]. There are three major breeds in Egypt: Rahmani, Ossimi, and Barki. Rahmani is distributed mainly in north of the Nile delta, Ossimi in mid Egypt, and Barki in western Mediterranean coastal region. Minor breeds like Saidi and Sohagi are located in south Egypt [2].

Reproduction is a complex process, and fecundity traits such as ovulation rate and litter size can be genetically regulated by many genes with small effects and sometimes also by single genes with major effects, called fecundity (Fec) genes [3]. Various major genes have been reported to affect prolificacy in sheep, which include three related oocyte-derived components, namely, bone morphogenetic protein receptor type 1B (BMPR1B), known as FecB on chromosome 6 [4]; growth differentiation factor 9 (GDF9), known as FecG on chromosome 5 [5]; and bone morphogenetic protein 15 (BMP15), known as FecX on chromosome X [5, 6].

GDF9 is an oocyte-derived growth factor in the transforming growth factor β (TGF-β) superfamily. It is highly expressed in the oocyte and has a pivotal influence on the surrounding somatic cells, particularly granulosa, cumulus, and theca cells [7]. Paracrine interactions between the developing oocyte and its surrounding follicular cells are essential for the correct progression of both the follicle and the oocyte. GDF9 is essential for the overall process of folliculogenesis, oogenesis, and ovulation and thus plays a major role in female fertility [8].

Eight different point mutations (G1–G8) have been identified in Belclare and Cambridge prolific sheep breeds [5]. There are four mutations in GDF9 gene, identified in different sheep breeds: high fertility (FecG^H) in Belclare and Cambridge breed, Thoka (FecG^T) in Icelandic breed [9], Embrapa (FecG^E) in Santa Ines breed [10], and the fourth not establish edits name and allele in Norwegian white sheep (Finnsheep) breed [11]. The mutation in the GDF9 gene increases the ovulation rate in heterozygous individuals, but in two out of four mutations, follicle development is disrupted in homozygous individuals resulting in infertility [12]. The FecG^H mutation causes increased ovulation rate in heterozygous ewes, while homozygous ewes are sterile. But FecG^E allele in homozygote state increases ovulation rate and litter size [13].

Sheep are large mammals which have many similarities to humans in terms of physiology. They are easy to handle and suffer from many diseases which affect humans. The use of animal models obtained through genetic manipulation has proven to be very useful for identifying many different diseases [14]. In Egypt, the genetic diversity of sheep in respect to these important economic genes has not been sufficiently studied. But, the sequence analysis and polymorphism of the BMP15 gene (exon 2) have been studied [15]. Results showed that FecX gene was monomorphic and disagreement with litter size; therefore, it is indispensable to survey other genes in order to establish the marker-assisted selection technique. This investigation was carried out to explore the presence of polymorphism in GDF9 gene (exon 1) using DNA sequencing and PCR-restriction fragment length polymorphism (RFLP) methods in these Egyptian sheep breeds that can act as a marker for twinning and is helpful in breeding selection for genetic improvement programs.

Materials and methods

Experimental animals

The Saidi and Ossimi sheep breeds used in this study were selected based on their single/twin production in three repetitive production cycles. The data of 24 Saidi and 13 Ossimi ewe sheep were collected from different farms belonging to the Ministry of Agriculture, Egypt. These data were used to study the reproduction traits, i.e., lambing rate (%) and litter size. Twenty-four Saidi individuals (12 ewes which produce twins and 12 which produce single) and 13 Ossimi individuals (7 ewes which produce twins and 6 which produce single) were used to study the polymorphism.

Blood sampling

Whole blood samples (5 ml) were collected from the jugular vein of each 24 Saidi and 13 Ossimi ewes in vacationer glass tubes containing EDTA (1 mg/ml). Blood samples were transferred to the laboratory in an ice box kept at 4 °C until used. The experimental procedures were performed according to protocols approved by the Biological Studies Animal Care and Use Committee of Egypt. All efforts were made to minimize any discomfort during blood collection.

DNA extraction

Genomic DNA was extracted from 150 μl blood samples using xanthogenate protocol described by Tillett and Neilan [16]. The quantified DNA was stored at −20 °C until further processing.

PCR amplification of GDF9 gene

Specific primers 5′-GAATTGAACCTAGCCCACCCAC-3′ and 5′-AGCCTACATCAACCCATGAGGC-3′ were used to amplify GDF9 gene (exon 1), which corresponded to the GenBank accession number AF078545, according to Hanrahan et al. [5]. The primer was synthesized by Invitrogen, Biotechnology Co. Ltd. (USA). The PCR amplification was performed in 25 μl total volume, each PCR reaction mixture containing 12.5 μl Master Mix (OnePCR™), 1 μl of each primer, 2 μl of genomic DNA (50 ng/μl), and 8.5 μl of sterile deionized water. PCR conditions were as follows: an initial denaturation step at 94 °C for 5 min, 35 cycles of 94 °C for 1 min, 62 °C for 1 min and 72 °C for 2 min, and a final extension step at 72 °C for 10 min using thermal cycler 2720 (Applied Biosystems, USA). PCR products were checked by electrophoresis using 1.8% agarose gel in 1× TAE buffer. The products were then purified using the QIAquick Gel Extraction kit no. 28706 (QIAGEN) following the manufacturer’s instructions and sequenced by automated DNA sequencing reactions, which were performed using a Sequencing Ready Reaction Kit (Life Technologies) in conjunction with ABI-PRISM and ABI-PRISM Big Dye Terminator Cycler.

DNA sequence and phylogenetic analysis

A consensus sequence of GDF9 fragments from both Saidi and Ossimi ewes which produce twins and single was constructed by using the SeqMan™ II 4.05 package for Windows 32. These sequences were subjected to alignment with GDF9 sequences of the GenBank, EMBL, DDBJ, and PDB from breeds of Ovis aries using the BLASTN 2.2.18 and BLASTP 2.2.18 (Basic Local Alignment Search Tool) algorithm at http://www.ncbi.npm. The MEGA version 5.2 program was used to generate a phylogenetic tree using the unweighted pair group method with arithmetic mean (UPGMA) method according to Sneath and Sokal [17]. The evolutionary distances were computed using the maximum composite likelihood method [18].

Polymorphism detection, genotyping, and association analysis

Thirty-seven PCR products of partial GDF9 gene (exon 1) were digested using Pst1 and Msp1 restriction enzymes (Fermentas, Germany, #ER0611 and ER0541, respectively) according to the manufacturer’s instructions. A final reaction volume of 32 μl contained 10 μl PCR product, 18 μl H₂O free of nuclease, 2 μl of 10× buffer, and 2 μl (5 units) of each restriction enzyme. The final volume of the mixture was mixed gently and spun down for a few seconds and then incubated for 18 h at 37 °C in water bath and stopped at 65 °C for 10 min. Restriction digestion products were checked by electrophoresis using 3% agarose gel in 1× TAE buffer and staining with ethidium bromide. The 100-bp ladder was used as a molecular size marker. The digestion PCR products were analyzed, and moreover, the allele and genotype frequencies of GDF9 gene were determined.

Statistical analysis

Data were statistically analyzed using the SPSS program, version 16.0 [19]. Means of lambing rate percentage and litter size of each breed were compared for main effects and their interaction by Duncan’s multiple range test [20], when significant F values were obtained (P < 0.05). The coefficient of variance (CV) percentage was calculated for each breed individually according to the formula $\mathrm{CV}=\frac{S}{X}\times 100$.

Results

Reproduction traits

Fertility traits have a major impact on efficiency and profitability in lamb meat production. In this respect, the lambing rate and litter size of ewes which produce single and twin lamb from Saidi and Ossimi sheep breeds, respectively, reflected ovulation rate, which is an important economic value. The lambing rate percentage and litter size showed a significant difference between ewes which produce single and twin lamb in each Saidi and Ossimi sheep breed (Table 1), while the CVs of the Saidi and Ossimi breeds for lambing rate percentage are 13.29 and 2.42 and also for litter size are 11.36 and 1.68, respectively (Table 1).

Table 1.

Least squares means and coefficient of variance percentage for lambing rate and litter size of Saidi and Ossimi sheep breeds

Traits			CV (%)
	Producing single lamb	Producing twin lamb
Saidi ewes
Lambing rate (%)	79.43 b	88.33 a	13.29
Litter size	1.21 b	1.42 a	11.36
Ossimi ewes
Lambing rate (%)	81.50 b	84.00 a	2.42
Litter size	1.12 b	1.15 a	1.68

Values in the same row with different letters differ significantly (P < 0.05). Lambing rate (%) = (Number of ewes lambing / Number of ewes mated) × 100. Litter size = Total number of lambs birth / Number of ewes lambing. Coefficient of variance percentage = (S/X) × 100

Sequencing of GDF9 gene (exon 1) and phylogenetic analysis

A single fragment of approximately 710-bp nucleotide sequences was amplified from each ewe individual (24 Saidi and 13 Ossimi) sheep breads (Fig. 1). Alignments of two sequences from Saidi ewes that produce single or twin lamb revealed 99.86% similarity between them and base substitution A to T comparing Saidi ewes that produce single lamb with Saidi ewes that produce twin lamb, whereas alignments of two sequences from Ossimi ewes that produce single or twin lamb revealed 100% similarity between them.

Fig. 1.

PCR amplification of partial exon 1 of GDF9 gene from Egyptian sheep breed. a, b From Saidi sheep breed. c From Ossimi sheep breed. M 100-bp DNA ladder

The topology of UPGMA tree of Saidi and Ossimi sheep breads with six accession numbers of O. aries in the GenBank database was represented in a monophyletic group (Fig. 2). The DNA sequences of GDF9 gene successfully grouped Egyptian breeds and O. aries sheep into two main clusters. The first cluster is extremely diverse and consisted of four accession numbers (KT853039, KR063137, NM1142888, and FJ429111). The second cluster had Egyptian sheep breeds and closely related with one accession number (AF078545), whereas accession number (HE866499) was the most distant. Multiple sequence alignment between Egyptian sheep breeds and accession number AF078545 showed that the nucleotide no. 1727 (T) was similar in all Ossimi sheep breeds and Saidi ewes that produce twin lamb and changed to (A) in Saidi ewes that produce single lamb. This base substitution (A–T) maybe in the 5′-regulatory region of sheep GDF9 gene.

Fig. 2.

UPGMA dendrogram of ten Ovis aries sheep generated based on Sneath and Sokal distances. Branch lengths are shown above the branches of clades. Saidi breed 01: ewes which produce twin lamb, Saidi breed 02: ewes which produce single lamb, Ossimi breed 01: ewes which produce twin lamb, and Ossimi breed 02: ewes which produce single lamb

RFLP analysis and genotyping

The PCR-RFLP approach has been used previously to genotype prolific sheep [21]. The PCR products of GDF9 gene (exon 1) digested by Pst1 or Msp1 restriction enzymes were used to survey molecular marker for twinning. In the present study, Saidi and Ossimi sheep breeds were screened for Pst1 enzyme digestion (Fig. 3); the results revealed a monomorphic type of restriction pattern consisting of two bands with 710 and 400 bp in Saidi or Ossimi sheep breeds. On the other hand, Msp1 enzyme digestion revealed two types of restriction pattern (Figs. 4 and 5). In Saidi sheep breed, polymorphic restriction pattern consisted of one band with 710 bp for ewes producing single lamb and two bands with 710 and 600 bp for ewes producing twin lamb (Fig. 4). In Ossimi sheep breed, monomorphic restriction pattern consisting of one band with 710 bp was observed in ewe producing single or twin lamb (Fig. 5).

Fig. 3.

Digestion product of partial exon 1 of GDF9 gene from Saidi or Ossimi sheep breeds with Pst1 restriction enzyme. Lanes 1–4: single-producing ewe, lanes 5–9: twin-producing ewe, M: 100-bp DNA ladder

Fig. 4.

Digestion product of partial exon 1 of GDF9 gene from Saidi breed with Msp1 restriction enzyme. Lanes 1–4: single-producing ewe, lanes 5–9: twin-producing ewe, M: 100-bp DNA ladder

Fig. 5.

Digestion product of partial exon 1 of GDF9 gene from Ossimi breed with Msp1 restriction enzyme. Lanes 1–3: single-producing ewe, lanes 4–9: twin-producing ewe, M: 100-bp DNA ladder

The allele and genotype frequencies of GDF9 gene based on Msp1 restriction enzyme digestion are presented in Table 2. The allele frequencies are 0.73 and 0.27 in Saidi sheep breed and 1.0 and 0.0 in Ossimi sheep breed for Fec ⁺ and Fec ^G, respectively. The Saidi and Ossimi sheep genotype frequencies Fec ⁺⁺, Fec ^+G, and Fec ^GG were 0.533, 0.394, and 0.073 and 1.0, 0.0, and 0.0 according to the results of Msp1 enzyme, respectively (Table 2).

Table 2.

Allele and expected genotype frequencies of GDF9 in Saidi and Ossimi sheep breeds

Restriction enzymes	Sheep breed	No. of ewes	Expected genotype frequencies			Allele frequencies
			++	+G	GG	+	G
Msp1	Saidi	24	0.533	0.394	0.073	0.73	0.27
	Ossimi	13	1.0	0.0	0.0	1.0	0.0

Discussion

Many genes and their functions are highly conserved throughout the animal kingdom. It’ is very similar between species, including cell proliferation, metabolism, and growth regulation. This homology across species is a key to considering the possibility of studying diseases and their underlying molecular mechanisms in animal models of genetic disease. Indeed, animal models have been crucial in understanding both genetic diseases [22] and non-genetic diseases [23]. Sheep are relatively outbred and much more human-like than mice, due to their closer genetic and physiological composition [24].

GDF9 is expressed in oocytes and is an attractive candidate gene for primary ovarian insufficiency (POI) because it is, like BMP15, a member of the TGF gene family. Increased frequencies of certain novel variants have been detected in European, Caucasian, and Asian patients [25–27], but not in Japanese and New Zealand populations [28–30].

Ovulation is a complex mechanism that differs among species and depends both on genetic and environmental factors. Mammals can be either mono- or polyovulatory based on how many oocytes mature and are released during ovulation. Ruminants typically release a single oocyte per ovulation compared to pigs and rodents which have high ovulation rates [31]. The ovulation rate even differs between breeds. In sheep, it ranges from one egg per ovulation in Texel and Suffolk to ten eggs per ovulation in the prolific Booroola Merino breed [4, 32]. Litter size differs between and within sheep breeds, and it depends on ovulation rate and is affected by the number of fertilized oocytes. Our results showed that litter size differs between and within sheep breeds and in accordance with those obtained [9]. The genetics of sheep litter size has been investigated by many researchers [5, 33–35]. The GDF9 gene, also called FecG, is located on chromosome 5 and codes for oocyte-derived GDF9 and is essential for normal folliculogenesis. The growth factor is present in oocytes from the primary stage of follicular development until ovulation [5]. Multiple genes were identified having substantial effects on reproduction traits, and some of them are most important being affecting prolificacy in animals. High litter size is an economically important trait that enhances sheep productivity in terms of producing a higher number of lambs, meat, and wool [33].

Bodensteiner et al. [36] reported the nucleotide sequence of the ovine GDF9 gene (GenBank accession number AF078545). Ovine GDF9 spans approximately 2.5 kb and contains two exons and one intron. Exon 1 extends from 1780 to 2178 bp, while exon 2 extends from 3302 to 4266 bp. Multiple sequence alignment between Egyptian sheep breeds and accession number AF078545 showed that the nucleotide No. 1727 (T) was similar in all Ossimi sheep breeds and Saidi ewes that produce twin lamb and changed to (A) in Saidi ewes that produce single lamb. This base substitution (A–T) maybe in the 5′-regulatory region of sheep GDF9 gene. In sheep, polymorphic sequence variations have been identified in GDF9 gene. Nucleotide changes (G-A) in G1 allele [5], (C-T) in FecG^H allele [5], (A-C) in FecT^T allele [12], (T-G) in FecG^E allele [13], (C-T) in FecG^V allele [37], and (G-A) [11] were identified. One nucleotide transition mutation (A125G) and one 5-bp deletion/insertion (TTCTT) mutation at the 163–167 locus in 5′-regulatory region of sheep GPR54 gene were identified in Small Tail Han and Corriedale sheep [38]. The transition mutation did not have a significant effect on prolificacy, but deletion mutation (particularly C nucleotide) seemed to have a significant effect on prolificacy.

RFLP is a technique in molecular biology to differentiate minor nucleotide sequence variations in homologous fragments of DNA. The technique relies on the specificity of restriction endonucleases, which are highly sequence-specific and cut the double-stranded DNA only at their recognition sites. The action of restriction enzymes produces variable lengths of homologous DNA molecules containing minor sequence variations or polymorphisms, and the cleaved fragments in the digested DNA can be separated by electrophoretic techniques. RFLP has a good repeatability and stability. The polymorphisms of three candidate genes in five Egyptian and Saudi sheep breeds (Barki, Ossimi, Rahmani, Najdi, and Harri) were detected by PCR-RFLP. The results showed that the polymorphism frequencies of FecB gene with Avall digestion are significantly imbalanced in five breeds [39]. The PCR-RFLP approach has been used previously to genotype prolific sheep [21]. The presence of one copy of GDF9 gene increases the fecundity rate in Saidi sheep and the same result reported by Hanrahan et al. [5], Juengel et al. [40], Liao et al. [41], and Ala Noshahr and Rafat [42]. This indicates that the presence of one copy of mutant GDF9 gene increases fecundity rate in sheep; the present study showed the same result reported by Hanrahan et al. [5], Davis et al. [9], Juengel et al. [40], and Liao et al. [41]. Ewes heterozygous for GDF9 mutations have increased ovulation rates, whereas homozygous ewes are sterile due to a failure of normal ovarian follicular development [5, 6]. Generally, many different loci effect reproduction and ovulation rate between different breeds of sheep, more than genetic background, are under control of age, season, and nutrition. According to these and the high prolificacy in these breeds, it is concluded that high prolificacy may be under control of other factors such as age, season, and nutrition or maybe there is another major gene in Egyptian sheep. Ewes heterozygous for GDF9 have increased lambing rate and litter size, whereas wild-type ewes had reduced ovulation rate. On the other hand, analysis of polymorphism for GDF9 (FecG^H) loci in Shal sheep indicates that the genetic factor responsible for twinning or multiple lambing rates is not related to the reported mutated alleles at the GDF9 major gene in this breed [43].

In conclusion, there are fecundity genes with major effect on ovulation rate and litter size in different sheep breeds. The FecG gene was polymorphic and in agreement with the litter size to establish marker-assisted selection method. The knowledge of fecundity genes is important to understand the process of fertility and infertility in mammals and thereby be able to treat genetic disorders associated to reproduction.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This study has been carried out in accordance with EU directive 2010/63/EU for animal experiments (http://ec.europa.eu/environment/chemicals//lab_animals/legislation_en.htm ).