Polymorphisms of ESR1, PGR and CYP19A1 Genes and their Association with Litter Size in Small-Tailed Han Sheep

1Key Laboratory of Animal Genetics and Breeding and Reproduction of Ministry of Agriculture and Rural Affairs, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China 2College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China. Article Information Received 18 January 2022 Revised 05 April 2022 Accepted 23 April 2022 Available online 08 September 2023 (early access)


INTRODUCTION
M ost of the sheep belong to seasonal estrus single lamb breed, only a few sheep belong to multiple lamb breed and its distribution is limited by region, the contribution of the number of lambs to the economic benefits of sheep breeding can reach 74% to 96% (Notter, 2012). Reproductive character is an important economic character of sheep. At present, there have been many research O n l i n e

F i r s t A r t i c l e
studies have shown that ESR1 is associates with in obesity, ovarian and breast disease in women (Guclu-Geyik et al., 2020;Turner et al., 2020;Tan et al., 2020). Progesterone receptor (PGR), also known as NR3C3, is a member of the steroid hormone nuclear receptor family (Arck et al., 2007;Tsai and O'malley, 1994). Previous studies have shown that PGR is highly expressed in ovary and uterus of Smalltailed Han (STH) sheep and can be used as a candidate gene for multiple lambs in sheep (Tian et al., 2018). In addition, CYP19A1 many candidates first exons and nine different transcription start sites are known, which can carry out complex and tissue-specific regulation (Bouchoucha et al., 2014;Grumbach and Auchus, 1999). Aromatase have been described, which are products encoded by CYP19A1 gene. In vertebrates, it can catalyze synthetases of different forms of sex hormones, which are also key enzymes in the process of rate limiting estrogen synthesis. The level of its activity and the high and low levels of estrogen in the synthesis reaction are widely distributed. It is involved in the production, processing, and transportation of protein in various tissues and cells of animals. It can not only act on the reproductive system, but also affect the occurrence of animal tumors (Balthazart and Ball., 1998;Ghosh et al., 2009). Therefore, the research of CYP19A1 is crucial for sheep reproduction. At present, CYP19A1 gene has been mostly reported in mice, chicken, human and other animals, but there are few studies on sheep breeding and reproduction (Zhang and Xu, 2021;Jin et al., 2020;Szaflik et al., 2020). Small-tailed Han sheep (STH) is famous with the early maturity, good meat quality, genetic stability and perennial estrus (Jiao et al., 2020), so that, the research on litter size has been a hot topic. In this study, major genes or genetic markers related to litter size of sheep were found. To identify DNA markers for selecting elite sheep at an early stage through marker-assisted selection (MAS), Sequenom MassARRAY ® SNP assay and detection of single nucleotide polymorphisms (SNPs) in four loci of ESR1, PGR and CYP19A1 genes in seven sheep breeds by DNA sequencing. The association of identified SNPs with the litter size performance traits of something was explored and the correlation analysis of different sheep population and STH sheep litter size at this locus can provide the basis for sheep breeding.

Ethics
All experimental procedures involving animals used in this study were approved by the Science Research Department (in charge of animal welfare issues) of the Institute of Animal Science, Chinese Academy of Agricultural Sciences (IAS-CAAS; Beijing, China). Ethical approval on animal survival was given by the animal ethics committee of IAS-CAAS (No. IAS2020-63, 28 April 2020).

Animals, sample collection and DNA extraction
Among this study, the seven sheep breeds were selected and divided into polytocous groups (Cele Black sheep; Hu sheep; and STH sheep) and monotocous groups (Prairie Tibetan sheep; Suffolk sheep; Sunite sheep; and Tan sheep) (Table I). Then, Jugular vein blood samples (10 mL blood per ewe) were collected using citrate glucose as an anticoagulant. Genomic DNA was extracted by the phenol-chloroform method, dissolved in ddH 2 O and stored at -20℃. At the same time, the lambing order and lambing number of STH sheep were collected.

Genotyping
First, single-base extended primers for g.75521224T> A, g.75378892A> T in ESR1, g.7491179C>T in PGR and g.56122588G>A in CYP19A1 were selected for genotyping in 768 samples from STH, Tan, Sunite, Suffolk, Hu, Cele Black and Prairie Tibetan sheep. Genotyping was performed using a MassARRAY ® SNP analysis (Johansen et al., 2013). The polymerase chain reactions system and temperature were described in detail in previous study . The DNA required quantity of each sample was 20μL, and the DNA concentration was 40-80 ng/μL.

O n l i n e F i r s t A r t i c l e
Statistical analysis Genotype frequencies, allele frequencies, heterozygosity (He), the number of effective alleles (Ne), p-values, and polymorphism information content (PIC) were calculated using the data obtained from genotyping results. Sheep populations with p > 0.05 (chi-square test) were considered to conform to the Hardy-Weinberg equilibrium (HWE). Multiple comparisons of means were performed using the least significant difference method. The applied model was expressed as follows: y ijn =μ+ P i + G j + IPG + e ijn , where y ijn is the phenotypic value of litter size; μ is the population mean; P i is the fixed effect of the ith parity (i = 1, 2, 3); G j is the fixed effect of the jth genotype (j=1, 2, 3); IPG is the interaction effect of parity and genotype; and e ijn is the random residual.

Protein structure prediction
The secondary structure of ESR1 with and without missense mutants was analyzed using SOPMA (https:// npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_ sopma.html). The STRING database (v.11.0) was applied to collect and integrate these functional interactions by consolidating protein-protein association data for massive organisms.

Polymorphisms of the ESR1, PGR and CYP19A1 genes
The genotyping results of the g.75521224T > A, g.75378892A > T, g.7491179C >T, and g.56122588G > A loci of ESR1, PGR and CYP19A1 are shown in Figure  1. For example, for g.75521224T > A, there are three genotypes: wild homozygous TT, heterozygous AT, and mutant homozygous AA.

Population genetic analysis of SNPs in ESR1, PGR and CYP19A1
Four SNPs (g.75378892A >T, g.75521224T > A) in ESR1, (g.60495375A >G) in PGR and (g.56122588G>A) in CYP19A1 were detected in monotocous and polytocous sheep breeds (Table II). The genotype frequency and allele frequency of g.75521224T > A locus were significantly different between polytocous and monotocous sheep (p < 0.05); and the genotype frequency and allele frequency of g.7491179C > T locus in PGR and g.56122588G > A locus in CYP19A1 were extremely significantly different between polytocous and monotocous sheep (p < 0.01) ( Table I).
The genotype frequency, allele frequency, He, Ne, PIC and χ2 test results from population genetic analysis for four SNPs in the seven sheep breeds are listed in Table III. The results showed that two (g.75378892A > T, g.75521224T > A) loci in ESR1 were at moderate polymorphism (0.25 < PIC < 0.5) in STH, Suffolk, Sunite, Cele Black, Hu, Tan, and Prairie Tibetan sheep; g.7491179C > T in PGR locus had low polymorphism (PIC < 0.25) in seven sheep populations; g.56122588G > A locus in CYP19A1 was at moderate polymorphism (0.25 < PIC < 0.5) in Suffolk, Cele Black, Hu, Tan, and Prairie Tibetan sheep except for STH sheep, Sunite (PIC < 0.25). In addition, the χ2 test revealed that g.75521224T > A was in Hardy-Weinberg equilibrium (HWE) (p < 0.05) except for STH sheep and Suffolk sheep; the g.75378892A > T was in HWE in the seven sheep breeds (p > 0.05); the g.7491179C > T was in HWE in the six sheep breeds (p > 0.05) except for Suffolk sheep; g.56122588G > A locus was under HWE (p >0.05) except for Hu sheep.

Association between four loci in ESR1, PGR and CYP19A1 with litter size in STH sheep
The results revealed that the g.75378892A > T locus in ESR1 was significantly correlated with litter size in STH sheep and that the litter size of ewes with AA genotype was higher than that of ewes with the AT and TT genotype (p < 0.05). However, g.75521224T > A, g.7491179C > T, g. 56122588G > A loci were not significantly correlated with litter size in STH sheep (p >0.05) (Table IV).  Bioinformatic analysis of ESR1, PGR and CYP19A1 Bioinformatics analysis showed that g.75378892A > T was A missense mutation, leading to the mutation of isoleucine at 36 to phenylalanine, which may cause some changes in the function of this secreted factor in follicles. The results were identified of alpha helix, extended strand, random coil, beta turn was changed, and many proteins interact (Figs. 2 and 3).

Relationship between ESR1 polymorphism and reproductive performance of animals
Combined with estrogen, ESRα plays an important role in the growth and development of embryonic mammary glands and female follicles during the reproductive cycle . Knockout of ESR1 gene would lead to disorders of LH hormone regulation and ovulation failure of ovaries in mice (Hewitt and Korach, 2003). In addition, many studies have confirmed that ESR gene is one of the major genes regulating high litter size in pigs (Findlay et al., 2001). Wang et al. (2015) used genome-wide association analysis to find that the T669C mutation of ESR1 gene  Note that different small letters in the same group mean significant difference (p < 0.05).
was closely related to the litter size of pigs and found that ESR1 gene was involved in the process of pig domestication. Studies of STH sheep and pigs have shown that ESR gene is closely related to lamb multiplication (Bi et al., 2005;Rahman et al., 2021). That is, more and more evidences indicate that mutations in the ESR1 gene locus are related to animal reproduction. We found that the ESR1 gene g.75378892A > T and g.75521224T > A loci were moderately polymorphic in seven sheep populations (0.25 < PIC < 0.5). Except that g.75521224T > A locus were not HWE in STH sheep and Suffolk sheep, the remaining SNPs showed HWE in different sheep breeds. This could be due to an imbalance in the site due to natural selection or artificial intervention, or it could be due to the small number of sheep involved in this study. Association analysis showed that the polymorphism of ESR1 in g.75378892A > T was significantly associated with different litter size of STH sheep (p < 0.05). Litter size of AA type ewes was significantly higher than that of TT type ewes (p < 0.05). Therefore, in the process of sheep breeding, AA homozygous individuals can be selected and retained to improve the fertility of sheep. In addition, bioinformatics analysis showed mutations in ESR1 in g.75378892A>T sites, resulting in amino acid changes, which we speculate is the main reason for the decline in lambing in STH sheep.

Relationship between PGR polymorphism and reproductive performance of animals
Studies have shown that PGR are low expressed in all major physiological systems of the animal body, but high expressed in the central nervous system and female reproductive tract (Pelch et al., 2011). Song (2017) obtained the polymorphism information of PGR gene through DNA mixed pool sequencing and flight mass spectrometry typing. According to the correlation analysis of PGR with Rex rabbit reproductive performance, it was found that the three SNPs of PGR were significantly correlated with the live litter size, weaning number and litter weight at 21 days of age. There is a significant association between the PGR rs660149 G variant of the Malays and the susceptibility to preterm birth (Langmia et al., 2015). The expression of PGR related genes affects piglet size, such as IHH, NR2F2 and BMP2 (Chen et al., 2016). In this study, it was found that the PGR gene g.7491179C > T was low polymorphism (PIC < 0.25) in all seven sheep breeds and had no significant correlation with litter size of STH sheep. Previous studies have shown that mutations in the PGR gene are associated with mammalian reproduction, which is inconsistent with this study and may be a species difference.

Relationship between CYP19A1 polymorphism and reproductive performance of animals
The aromatase encoded by the CYP19A1 gene is an enzyme for estrogen synthesis, a monooxygenase, which can catalyze many reactions related to steroids and catalyze the conversion of androgens to estrogen (Bershteĭn, 1997). Cytochrome P450 aromatase is involved in the development of follicles at different stages and has a regulatory role (Bao and Garverick, 1998) and plays an important role in the development of sheep gonadal (Kwon et al., 2001). The development of testicular cells (Hess and Roser, 2004) is also very important. Previous studies on the CYP19A1 gene have mainly focused on human cancer-related diseases (Wang et al., 2021;Akçurin et al., 2016) and the reproductive regulation of other species, and there has been little research in the field of O n l i n e

F i r s t A r t i c l e
Polymorphisms of ESR1, PGR and CYP19A1 Genes and their Association with Litter Size sheep reproduction. Moreover, Zhang and Xu (2021) used Yulin mixture to increase the expression of CYP19A1 in mice, thereby regulating the proliferation and apoptosis of granulosa cells. Taking the STH sheep population with different fecundity as the research object, it is found that the CYP19A1 gene is mainly expressed in the ovary and hypothalamus tissues. The expression level of the CYP19A1 gene in the ovary tissues of the multi-lamb STH sheep is higher than that of the single-lamb and Sunite sheep (p < 0.05) (Tian et al., 2019). Vega et al. (2018) found that the rs718446508T > C and rs41651668T > C sites of the CYP19A1 gene were significantly related to bovine oocyte production, survival, embryonic development, pregnancy and other reproductive traits (p < 0.05). El-Bayomi et al. (2018) found that the CYP19A1 gene c.135T > C, c.559G>A, c.1285C > T, c.1394A > G sites were significantly related to the buffalo interestrous period (p <0.05). The results of the correlation analysis showed that there was no significant correlation between the different genotypes of CYP19A1 gene g.56122588G>A and the litter size in different parities of STH sheep (p > 0.05). It may be because natural selection or artificial selection has a greater impact on the distribution of the site, or it may be related to the number of sheep breeds we have selected. In addition, it may be that the site is in the 5'UTR region and does not directly participate in the encoding of the protein, thereby affecting its function.

Protein secondary structure prediction and interaction
Interestingly, through the construction of protein network interaction maps, interactions between the ESR1, PGR and NCOA1 genes were found. The NCOA1 gene, known as the steroid receptor co-activator protein 1 (SRC1) gene, is an estrogen receptor, progesterone receptor assistive activator (Oñate et al., 1995) and is considered a "switch" of reproductive hormones (Seitz et al., 2008) Breeding in chickens (Gholami et al., 2014) and sheep (Yuan et al., 2019;Xu et al., 2018) has been reported in many studies and has achieved more desirable results. However, the results of this study showed that the mutation of A>T site in g.75378892A resulted in the decrease of litter size of small-tailed Han sheep. It could be speculated that the mutation of this site reduced or blocked the recognition of ESR1 and NCOA1 genes, thus affecting the reproductive rate. In addition, the PGR site is synonymous mutation, which does not cause the secondary and tertiary structure mutation of the protein, so it cannot interact with NCOA1, so it has no significant influence on lambing of small-tailed Han sheep. CYP19A1 also interacts with many proteins, and whether it affects reproduction needs to be further explored.

CONCLUSION
In this study, we first performed a population genetic analysis of ESR1, PGR and CYP19A1 SNPs. Through association analysis, we found a key gene locus, namely g.75378892A > T in ESR1, this locus could influence each parity litter size in Small-tailed Han sheep (p < 0.05). Therefore, our studies could be useful in marker-assisted selection of the litter size in Small-tailed Han sheep.