FSHR (EXON 10) GENE POLYMORPHISMS AND ITS ASSOCIATION WITH FERTILITY TRAIT IN EGYPTIAN

For the association between of Follicle stimulating hormone receptor (FSHR) gene (partial part of exon 10) polymorphisms and litter size trait in Egyptian Ossimi sheep, polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP) and DNA sequencing techniques were developed. Fifty female Ossimi sheep reared under Egyptian conditions were selected according to their litter size. DNA from blood samples of these animals was isolated to amplify 250-bp of the FSHR gene influencing litter size production trait in sheep. Based on litter size, 50 animals were selected from the highest to the lowest litter size productivity during three seasons. PCR-SSCP analysis of the FSHR gene (250-bp) showed two various genotypes AA and AB with frequencies 0.64 and 0.36, respectively. The frequencies of the A and B alleles were 0.82 and 0.18, respectively. PCR fragment of FSHR gene (191-bp) was sequenced only in the high and low litter size productivity animals (GenBank accession numbers from MG973191 to MG973207, sequentially). The result indicated that 6SNPs (G/71, G/72, G/77, A/110, A/111, A/191) in high fertile animals, while, 10 SNPs (T/1, C/2, T/14, A/69, A/70, A/71, A/74, G/74, A/75, A/136) have found in low fertile animals. Statistically, AA and AB genotypes have no significant differences (p> 0.05) on litter size trait in Ossimi sheep. FSHR (exon 10) locus was moderate polymorphic (PIC= 0.25) and it can be used for high litter size productivity in Ossimi sheep as a marker-assisted selection (MAS).


Introduction
Sheep (Ovis aries) have positive economic benefits for human. Where, this kind of animals is preferable as a result of low maintenance cost, adaptation in many environments in the world and resourcefulness for many products such as meat, milk, wool and hides (Abulyazid et al., 2011). In Egypt, sheep meat production is more important than fiber production (6% of the total animal meat production). Ossimi sheep (under study) is an Egyptian Nile Valley breed, which is one of the three major Egyptian sheep breeds besides Barki and Rahmani which represent 65% of the total population of sheep in Egypt (ICARDA annual report, 2007). Ossimi sheep is a most popular and environmentally adaptable in Lower Egypt. In addition, it is a carpet wool breed (Mason, 1996). The mean of Ossimi Kidding rate production under Egyptian condition is 1.22 (lamb/ewe) (Elshenawy, 1995).
Improvement o f fertility trait in sheep has become desiring interest for breeders, where moderate increases in litter size can equal outsized gains in profit (Abraham and Thomas, 2012). This can be achieved by improving the genetic worth of the stock by proper selection methods for improving reproduction rate and production efficiency. Therefore research community constantly searches some affecting genes that can act as marker influencing fertility in animals by marker assisted selection (Mishra, 2014). Fertility trait can be improved using marker assisted selection (MAS) which make improvement by detecting variations of fertility or fecundity genes linked with high and low fertile animals (Abdoli et al., 2016).
Many genes affect fertility in sheep such as Boroola gene, growth differentiation factor 9 gene and follicle stimulating hormone receptor gene. This study is concerned with Ovis arias's FSHR gene which located in chromosome 3 (3:75470485-75470694) in sheep (n=54). Sequence version Oar_v3.1 in www.ensemble.com has determined that chromosome 3 has 1994 coding genes and 523 noncoding genes, one of these coding genes is FSHR gene (size= 2088bp) consists of 10 exons coded into FSHR protein (656 amino acids). FSHR gene has a large number of variants which differentiated into two categories missense and synonymous. FSH (Follicle stimulating hormone) secreted by an anterior pituitary regulates gonadal functions in male and female, it is under regulation of gonadotropin releasing hormone (GNRH) as well which activates its receptor in granulosa cells (GCs) in the ovary.
FSHR is a member of the family of G-coupled protein activated FSH which secreted by anterior pituitary regulates gonadal functions in males and females, as well as, it is under regulation of gonadotropin releasing hormone (GNRH) (Richards and Midglay, 1976). Thus, the level and the target of hormone response are controlled by mechanisms that determine FSHR levels and cell specific expression, which are supported by transcription of its genes (George et al., 2011;Dias et al., 2002;Bogerd, 2007).
Exon 10 of FSHR gene is large and encodes the C-terminal part of the extra cellular domain (ECD) (hinge region), the transmembrane domain (TMD) and the intracellular domain of the receptor (Fan & Hendrickson 2005;Jiang et al., 2012).The objectives of the present study are to detect SNPs (mutations) in a partial region of exon 10 of FSHR gene in a high fertile Egyptian sheep breed (Ossimi sheep) using PCR-SSCP and sequencing techniques. Also, to investigate the relation between the FSHR gene and fertility trait in Ossimi sheep breed under Egyptian conditions.

Materials and Methods
Animals. Blood samples were collected from the jugular vein of 50Ossimi ewes (5ml/ewe) puncture into tubes containing an anticoagulant disodium EDTA. The samples have stored at -20°C until needed for DNA isolation. DNA isolation. Genomic DNA was isolated from whole blood samples using a commercially available kit (EZ-10 Spin Column Genomic DNA kit for Blood samples, Bio Basic, Canada). Isolated genomic DNA was separated on agarose gel electrophoresis using 1% (w/v) agarose in 0.5X TBE buffer. The gel was photographed using the gel documentation system (Syngene, UK) to visual genomic DNA band. PCR amplification and genotyping of FSHR gene. 250-bp fragment of exon10 of FSHR gene in 50Ossimi sheep was amplified by PCR using forward (5'-ATCACGCTGGAAAGATGGCATACC-3') and reverse (5'-ACATTGAGCACAAGGAGGGAC-3') primers . PCR was performed in a reaction volume of 25μl using 200ng of genomic DNA of each sample, 25 pmol of each primer and 2X Taq DNA polymerase Mix (Bioline, UK). Thermal cycling (Peltier-based Thermocycler, Long Gene) was carried out by initial denaturation at 94°C for 5 min, followed by 30 cycles each at 94°C for 1 min, annealing temperature at 60°C for 30 sec, polymerization temperature at 72°C for 30 sec and final extension at 72°C for 10 min, then the samples were held at 4°C. The amplified DNA fragments were separated on 2% agarose gel, stained with ethidium bromide, visualized on a UV Transilluminator and photographed by Gel Documentation system (Alpha Imager M1220, Documentation and Analysis System, Canada). Single stranded conformational polymorphism (SSCP). Aliquots of 5μlPCR products were mixed with denaturating solution (98% formamide, 0.025%xylene cyanol, 0.025% bromophenol blue and 10 mM EDTA) and incubated at 98°C for 10 min and then chilled on ice rapidly. Denaturated DNA was loaded on10% PAGE gel (10X 10 CM) in 1X TBE buffer and constant voltage 65V for 5hours.
For staining DNA bands and visualizing, the gel was stained with ethidiumbromide and photographed by using gel documentation system. Sequencing. DNA sequencing for purified FSHR amplicon had performed by Genetic Analyzer (Applied Biosystems, Hitachi, Japan). Where, the highest eight (2, 4, 5, 6, 7, 13, 16 and 17) and the nine of lowest (21,22,23,24,25,26,28,40 and 41) litter size animals were sequenced (one direction, forward) at Genetic Engineering and Biotechnology Research Institute, City of Scientific Research and Technological Applications, Alexandria, Egypt. Statistical analysis. For litter size, each ewe has three seasons' records (winter, spring and next winter). Random selection of three parities ewes was done and documented in Sakha farm, Agricultural Research Station, Kafer Elshiekh, Egypt. Polymorphisminformation content (homogeneity).PIC (polymorphism information content) is a measurement that gives an indication how much the locus is highly, moderate or poorly mutated by knowing the alleles frequencies of this locus: PIC= 1-Ʃ (p ij ) x 2, where, pij is the frequency of different ij th allele of studied locus.The calculation of PIC was processed by PIC Calculator (www.liverpool.ac.uk). Q-Q Plot.We put the assumption of litter size values of the 50 animals are normal distributed, so we should test this assumption by run the Q-Q plot using (IBM SPSS Statistics, Version 22, 2013). A Q-Q plot is a scatterplot created by plotting two sets of quantities against one another, one of it is litter size values of tested animals and normal disrupted expected value that the program put it. If both sets of quantities came from the same distribution, we should see the points forming a line that's roughly straight.
Independent t-test.The Independent samples t-test compares two genotypes means to determine whether they are significantly different or not. The two independent samples (AA and AB genotypes) are assumed to be drawn from populations with unequal variances (i.e., σ 1 2 ≠ σ 2 2 ), the test statistic t is computed as: t= Where: 1 = Mean of first genotype AA; 2 = Mean of second genotype AB; n 1 = Sample size (i.e. number of observations) of AA genotype; n 2 = Sample size (i.e., number of observations) of AB genotype; s 1 = Standard deviation of AA genotype; s 2 = Standard deviation of AB genotype, Least mean squares analysis.Least mean square test was used to examine the relationship between different genotypes and the litter size. The least squares means were used for multiple comparisons in litter size among the different genotypes. Analysis was performed using the general linear model procedure of (IBM SPSS Statistics, Version 22, 2013).The litter size trait of Ossimi ewes was analyzed using the following fixed effects model: Where: yijklm= phenotypic value of litter size; m= population mean; Ai= fixed effect of theith age of ewe (i= 3, 4, 5, 6, 7, 9, 13); KSj= fixed effect of the jth kidding season (j= 1, 2, 3); Pk= fixed effect of the kth parity (k = 1, 2, 3); Gl= fixed effect of the lth genotype (l = 1, 2, 3); eijklm= random residual effect of each observation.

Table 3. Reference codon, mutated codon, amino acids variation and synonymous/nonsynonymous variants
In this study, fifty Ossimi sheep animals were divided into two groups according to their litter size (1.2 lamb/ewe); high fertile group (litter size= 2, 1.6 or 1.3) and low fertile group (litter size= 1).For understanding the relationship between polymorphism in FSHR gene locus (part of exon 10) and fertility trait in Ossimi sheep, SSCP and sequencing techniques were used, and then this relationship was statistically analyzed. However, we found then this locus is moderate polymorphic (PIC=0.25) and has two alleles (A and B) and two genotypes (AA and AB) in all animals. AA genotype frequency was 0.64 and AB genotype was 0.36, hence, A allele frequency was 0.82 and B allele was 0.18.
For 191-bp FSHR fragment sequencing, we found 6 SNPs in high fertile animals (G/71, G/72, G/77, A/110, A/111, A/191). While in low fertile animals, we found 10 SNPs (T/1, C/2, T/14, A/69, A/70, A/71, A/74, G/74, A/75, A/136). Statistically, AA and AB genotypes have no significant differences (p>0.05) in litter size, as well as, regression statistics considered that 10% of genotype differences could describe the differences of litter size in this sample (fifty animals). The results have a valuable meaning in choosing this polymorphic locus of FSHR as a candidate gene for research and breeding programs of fertility trait. These results preliminary showed that FSHR gene is a major gene that influences the prolificacy of experimental animals or a molecular marker in close linkage with prolificacy trait. Consequently, FSHR gene has considered as a possible candidate gene for increasing litter size in Ossimi sheep.
In the previous related studies, FSHR gene polymorphisms and its association with litter size in animals classified to two categories according to regulatory and their translated regions of FSHR gene. An example for this, the 5' regulatory region of FSHR gene had a significant effect on fertility trait and proofed as a marker-assisted selection (MAS) in the improvement programs. Where, Chu et al.(2012)found that in 5' regulatory region of FSHR gene 2 mutations (-681 C/T and -629 C/T) in Hu sheep and 3 mutations (-200 G/A, -197 G/A and -98 T/C) in small tail Han sheep. In the same study, the author found that the heterozygous Small tail Han sheep (EG and EF) had 0.89 lambs more than the homozygous. In another study on sheep, Wang et al.,(2015) found that the CC genotype of FSHR gene in small tailed and Han sheep had lamb production more than those the TC and TT genotypes with 0.52 (p< 0.01) and 0.72 (p< 0.01), respectively.
The study in goat, Xiangdong Black, NanJiang Brown and Guizhou Black litter size was affected significantly by FSHR gene (p<0.05) only in Guizhou Black goat and BB genotype was significantly higher than AA and AB genotypes (Zhu et al., 2007). On the other hand, in these three goat breeds, the author found the same five SNPs (-93C/A, -80 G/C,-63 C/A, -56C/G and -55 T/C) of 5' regulatory region of FSHR gene which were not necessarily affect significantly on litter size. In another study in three goat breeds (Jining Grey, Beor and Inner Mongolia Cashmere), Guo et al.(2013) found three genotypes CC, CD and DD. Only in Jining Grey goat, the author noticed that the CC genotype had 0.46 and 1.3 kids more than the other CD and DD genotypes, respectively. While CD genotype had 0.57 kids more than DD genotype. On the other hand, the two transversions in 70T/A and 130G/C positions of amplified sequenced region of FSHR gene, are found in DD and CC genotypes in the three previous goat breeds. In Yunling Black and Boer goat breeds, Cui et al., (2009) found four mutations in the coding region of FSHR gene 486C/A (162Arg/Ser), 1042C/G (348Pro/Ala), 1930T/A (644 Phe/Ile) and 2036T/C (679Thr/Ile). In Yunling Black goat only, the author recognized that the FSHR mRNA and protein expression levels were significantly and positively correlate with fecundity, as well as reduction levels may be associated with the fewer observed oocytes and fewer follicles.
In cows (Bostaurus and Bosindicus), Marson et al., (2008) evaluated the effect of polymorphisms in exon 10 of FSHR gene using PCR-RFLP (AluI restriction enzyme) and the results showed three genotypes GG, GC and CC. Genotype GC revealed higher pregnancy rate (66%) than the two other genotypes (GG and CC). According to the author, has observed no significant effect of the three genotypes (GG, GC and CC) in both Bostaurus and Bosindicus beef populations. In another research, Yang et al., (2010) found a mutation (-278 G/A) and three genotypes (CC, CD and DD) in Chinese Holstien cows. Genotype CC had a significant increase in the total number of ova and transferable embryos than the other two genotypes, which had an absence of super ovulation response. Also, Sharifiazdi et al., (2018) emphasized that this mutation (-278 G/A) may affect some reproductive variables in Holstein dairy cows.
Lastly in Erhualian and Yorkshire sows, Zhang et al., (2002) detected a transition mutation (566 C/T) in exon 7 of FSHR gene. Consequently, a prediction of Ala/Val substitution has done at residue 189 in ECD and this polymorphism significantly associated with the total born number and number born alive. However, our results with the other previous mentioned studies confirm that the FSHR gene in Ossimi sheep will be a strong candidate gene for further applications in marker-assisted selection (MAS) and breeding programs for fertility trait improvement.

Conclusion
PCR-SSCP genotyping and DNA sequencing techniques were developed to study the association between FSHR gene polymorphism and fertility trait in fifty Ossimi sheep. The results indicated two genotypes (AA and AB) which have no significant differences (p> 0.05) on litter size trait, but this locus (partial part of exon 10) was moderate polymorphic (PIC= 0.25). Using DNA sequencing, in the high fertile animals 6 single nucleotide polymorphisms (SNP's) at 6 different positions have observed G/71, G/72, G/77, A/110, A/111, A/191. While in the low fertile animals, 10 SNPs (T/1, C/2, T/14, A/69, A/70, A/71, A/74, G/74, A/75, A/136) were observed. Thus, these findings can be used as marker-assisted selection (MAS) for high fertility trait in Ossimi sheep reared under Egyptian conditions.