Insertion/deletion variants within the IGF2BP2 gene identified in reported genome-wide selective sweep analysis reveal a correlation with goat litter size

Insertion/deletion variants within the IGF2BP2 gene identified in reported genome-wide selective sweep analysis reveal a correlation with goat litter size 已报道的全基因组选择性消除分析中发现 IGF2BP2 基因的插入/缺失变异与山羊产羔数相关 1 first-author 1 1 1 1 1 2 1 2 1 corresp Xianyong LAN, lanxianyong79@126.com Xianyong LAN, lanxianyong79@126.com lanxianyong79@126.com https://orcid.org/0000-0002-6104-2904 Lab of Animal Genome and Gene Function, College of Animal Science and Technology, Shaanxi Key Laboratory of Molecular Biology for Agriculture, Northwest A&F University, Yangling712100, China Lab of Animal Genome and Gene Function, College of Animal Science and Technology, Shaanxi Key Laboratory of Molecular Biology for Agriculture, Northwest A&F University, Yangling712100, China Life Science Research Center, Shaanxi Provincial Engineering and Technology Research Center of Cashmere Goats, Yulin University, Yulin719000, China Life Science Research Center, Shaanxi Provincial Engineering and Technology Research Center of Cashmere Goats, Yulin University, Yulin719000, China Insulin-like growth factor 2 mRNA-binding protein 2 ( IGF2BP2 , also called IMP2 ) plays an essential role in the development and maturation of germ cells and embryos and is a candidate gene for goat litter size, based on a previous genome-wide selective sweep analysis. In this study, the mRNA expression level of IGF2BP2 was found to be significantly higher in a single-lamb group than in a multi-lamb group. Insertions/deletions (indels) within the goat IGF2BP2 gene, including P4-Ins-13bp and P5-Del-12bp, were verified in 918 Shaanbei White Cashmere (SBWC) female goats. The minor allelic frequencies (MAFs) of P4-Ins-13bp and P5-Del-12bp loci were 0.349 and 0.295, respectively. Analysis using the Chi-square ( χ 2 ) test showed that the genotype ( χ 2 =14.479, P= 0.006) distribution of P4-Ins-13bp was significantly different between the single-lamb and multi-lamb groups. Correlation analysis demonstrated that P4-Ins-13bp was significantly associated with goat litter size ( P= 0.022), and individual goats with the homozygous deletion/deletion (DD) genotype produced more litters than other goats. Therefore, considered as a potential molecular marker significantly related to lambing traits, the P4-Ins-13bp mutation of the goat IGF2BP2 gene can be used in goat breeding with practical molecular marker-assisted selection (MAS) to optimize female reproduction and improve economic efficiency in the goat industry. 目的 2 验证 IGF2BP2 插入/缺失与陕北白绒山羊产羔数之间的关系，并选择有利的等位基因。 创新点 2 首次筛选、验证山羊 IGF2BP2 基因的InDel位点，确定其与山羊产羔性状的相关性及作用，为山羊经济性状改良及新品种培育提供理论依据。 方法 2 采用实时荧光定量聚合酶链式反应（qRT-PCR）方法分析 IGF2BP2 在单羔组和多羔组中的表达水平；通过PCR扩增-琼脂糖凝胶电泳在918只陕北白绒母山羊中鉴定 IGF2BP2 基因P4-Ins-13bp和P5-Del-12bp缺失/插入位点基因型，并进行测序以验证突变；利用SHEsis平台分析两个位点的连锁不平衡；采用方差分析（ANOVA）和 χ 2 检验分析山羊 IGF2BP2 基因变异与产羔数的关系；并利用在线数据库预测转录因子（TF）和突变区域序列的结合。 结论 2 本研究发现， IGF2BP2 的mRNA表达水平在单羔组明显高于多羔组。在陕北白绒母山羊中验证了 IGF2BP2 基因内的插入/缺失（indels），包括P4-Ins-13bp和P5-Del-12bp。 χ 2 检验表明，P4-Ins-13bp基因型（ χ 2 =14.479， P =0.006）在单羔和多羔组间分布差异显著；相关分析表明，P4-Ins-13bp与山羊产仔数显着相关（ P =0.022），具有缺失/缺失（DD）基因型的山羊个体产仔数高于其他山羊。因此， IGF2BP2 基因的P4-Ins-13bp突变可作为一种潜在的与繁殖性状显著相关的分子标记，以优化母羊繁殖力，提高山羊产业的经济效益。 Goat Insulin-like growth factor 2 mRNA-binding protein 2 ( IGF2BP2 ) Litter size Indel Marker-assisted selection (MAS) 山羊 IGF2BP2 产仔数 插入/缺失 分子标记辅助选择 1 Introduction 1 Litter size among domestic animals is a complex quantitative and economic trait and serves as a key factor in determining the economy of the goat-rearing industry ( Bünger et al., 2005 Bünger et al., 2005 ; Abdoli et al., 2019 Abdoli et al., 2019 ; Lu and Miller, 2019 Lu and Miller, 2019 ; Li et al., 2021 Li et al., 2021 ). To increase economic efficiency, improvements in goat reproductive quality should be prioritized. Marker-assisted selection (MAS), which is characterized as an alternative selection of DNA mutations, is not affected by the microenvironment. In addition, compared with traditional breeding techniques, MAS is a more accurate, efficient, and convenient genetic selection method ( Collard and Mackill, 2008 Collard and Mackill, 2008 ; Kang et al., 2019b Kang et al., 2019b ; Hui et al., 2020 Hui et al., 2020 ). However, identifying effective target markers associated with favorable outcomes remains necessary for practical application of MAS to the breeding process. In recent years, the rapid development of genome-wide selective sweep analysis and genome-wide association study (GWAS) have identified many candidate genes associated with the litter sizes of livestock, such as the porcine spermatogenesis-associated 19 ( SPATA19 ), ovine follicle-stimulating hormone receptor ( FSHR ), and bovine 2',‍5'-oligoadenylate synthetase 1 ( OAS1 ) ( Wang et al., 2012 Wang et al., 2012 ; Pan et al., 2014 Pan et al., 2014 ; Alex et al., 2018 Alex et al., 2018 ). In goats, previous studies have identified growth differentiation factor 9 ( GDF9 ), membrane-associated ring-CH-type finger 1 ( MARCH1 ), and DNA methyltransferase 3β ( DNMT3B ) as genes that affect fecundity ( Kang et al., 2019a Kang et al., 2019a ; Wang et al., 2019 Wang et al., 2019 ; Hui et al., 2020 Hui et al., 2020 ). A previous genome-wide selective sweep analysis performed in Dazu Black goats has identified insulin-like growth factor 2 (IGF2) mRNA-binding protein 2 ( IGF2BP2 , also called IMP2 ) as a candidate gene associated with litter size (E et al., 2019). IGF2BP2 is a member of the IGF2 mRNA-binding protein family (IGF2BP, also known as IMP), which includes IGF2BP1 , IGF2BP2 , and IGF2BP3 , and is a highly conserved post-transcriptional regulatory factor involved in RNA processing, localization, stability, and translation. The IMP family is expressed ubiquitously throughout the embryonic development process, and subsequently plays a crucial role during the normal growth and development of embryos ( Ruggiu et al., 1997 Ruggiu et al., 1997 ; Chennathukuzhi et al., 2003 Chennathukuzhi et al., 2003 ; Biswas et al., 2019 Biswas et al., 2019 ). Recently, one study demonstrated that 5-bp and 9-bp insertions/deletions (indels) in the IGF2BP1 gene were related to litter size during the second gestation in Australian White sheep ( Liu et al., 2021 Liu et al., 2021 ). It has been reported that the involvement of IGF2BP2 in the HMGA2-IMP2 pathway plays an important role in mouse embryonic development ( Brants et al., 2004 Brants et al., 2004 ). In addition, IGF2BP3 is highly expressed in human placental trophoblasts during early pregnancy and plays a vital role in embryo implantation and placenta formation ( Wang et al., 2016 Wang et al., 2016 ). IGF2BP3 is closely related to the RNA-binding protein (RBP), Vg1 RBP (also known as Vera), and is essential for mesoderm induction and left–right axis formation ( Li et al., 2014 Li et al., 2014 ). These data suggest that the IMP family may play key roles in follicular maturation and embryo development; however, thus far, whether mutations in the IMP family member IGF2BP2 affect goat reproductive traits and which genotype is advantageous remain unknown. To confirm the effects of IGF2BP2 polymorphisms on female goat reproductive capacity, we examined Shaanbei White Cashmere (SBWC) goats ( n =918) in this study. The primary aims of the study were to verify the relationship between indels in IGF2BP2 and SBWC goat litter sizes and to select the favorable alleles. We also wanted to lay a foundation for the application of MAS in goat breeding, to improve the economic efficiency of the goat livestock industry. 2 Materials and methods 1 2.1 Bioinformatics analysis 2 Nucleotide sequences from Capra hircus (XM_005675253.3), Ovis aries (XM_004003071.4), Bos taurus (NM_001192504.2), Sus scrofa (XM_021069998.1), Homo sapiens (NM_001007225.3), Mus musculus (NM_183029.2), and Gallus gallus (XM_015277139.2) were obtained from the National Center for Biotechnology Information (NCBI) database. Nucleotide sequence similarity was detected using the Basic Local Alignment Search Tool (BLAST) database (BLASTN) ( https://blast.ncbi.nlm.nih.gov/Blast.cgi https://blast.ncbi.nlm.nih.gov/Blast.cgi ), and the MEGA7 MUSCLE program was used to compare multiple nucleotide sequences and perform evolutionary analysis ( Kumar et al., 2016 Kumar et al., 2016 ). 2.2 Animal sample collection 2 Female adult goats of a representative local breed of SBWC ( n =918), which produce high-quality cashmere and meat, were randomly selected from a group with resistance to coarse feeding, wind, sand, and disease. Ear tissue samples were collected from healthy, unrelated adult female goats with similar ages. First-born litter sizes were recorded for 885 individuals. Ovarian tissue samples were collected from adult female goats, and three samples were collected from single-lamb (first-born litter size=1) and multi-lamb (first-born litter size > 1) groups maintained by an SBWC goat farm in Shaanxi Province, China. 2.3 DNA extraction, RNA isolation, and cDNA synthesis 2 DNA was extracted from the ear tissue samples using a previously described high-salt method ( Bi et al., 2020 Bi et al., 2020 ). The extracted DNA was stored at -80 ℃ and then diluted to a standard concentration (10 ng/µL), after which it was stored at -40 °C. The procedure used to isolate total RNA from ovarian tissue was described in a previous study ( Tang et al., 2021 Tang et al., 2021 ). After isolation, 1% (0.01 g/mL) agarose gel was used to verify RNA integrity. Complementary DNA (cDNA) was synthesized using a PrimeScript TM RT Reagent kit (TaKaRa, Dalian, China), according to the manufacturer's instructions. 2.4 Primer design and DNA pooling 2 Based on the goat IGF2BP2 (GenBank number: NC_030808.1) gene reference sequence obtained from the NCBI database and the variant table information obtained from the Ensembl database ( http://asia.ensembl.org/index.html http://asia.ensembl.org/index.html ), six potential indel loci were selected according to the length and location of the variants, and primers were designed using NCBI Primer Blast software ( Table 1 Table 1 ). A total of 30 DNA samples from SBWC individuals were randomly selected to construct a genomic DNA pool as the template for polymerase chain reaction (PCR) amplification to detect the presence of non-specific primer amplification, after which 50 random SBWC individuals were selected to detect the presence of indels at the six loci of the IGF2BP2 gene. F, forward; R, reverse; Ins, insertion; Del, deletion. Variant ID Locus name Primer sequence (5'→3') Product length (bp) (insertion/deletion) 2 rs655740526 2 P1-Ins-16bp F1: CTTCAAGGTATAATTCGCTGTGCAT 2 260/244 R1: GATATGCAACAAGCAACAAAGGT 2 rs637951471 2 P2-Del-14bp F2: TGATACTTGGGAGGGGATGT 2 197/183 R2: TGAAGCACATGTGGCAAAAGA 2 rs642405889 2 P3-Ins-13bp F3: GAATATTGGAAACAGCTGCTCAT 2 196/183 R3: AAAAGTTTGAAAATCCCACTCACA 2 rs660766378 2 P4-Ins-13bp F4: CTGTGGTCCAGACCTCCTCA 2 163/150 R4: TACAGACACTGTGAGAGGCG 2 rs655441618 2 P5-Del-12bp F5: CCAGTTCAGTGACCCATTCG 2 140/128 R5: TGCAAGAAGCATCTGTCTGCTA 2 rs649205677 2 P6-Del-6bp F6: GTGCCTAAGACTGTGTAATGCCA 2 208/202 R6: CTGGGATCAAACCTGGCACAT 2.5 Amplification, genotyping, and DNA sequencing 2 In this study, the PCR reaction volume (13 µL) and amplification steps (touch down-PCR) were the same as described ( Wang et al., 2020 Wang et al., 2020 ). The genotypes of different individuals were identified by 3% (0.03 g/mL)agarose gel electrophoresis, and sequencing was performed by Tsingke Biological Technology (Shaanxi, China) to verify mutations. 2.6 Quantitative real-time PCR 2 We analyzed the expression levels of goat IGF2BP2 in the single-lamb group ( n =3) and the multi-lamb group ( n =3) by quantitative real-time PCR (qRT-PCR). For this purpose, we used a 10-µL reaction system in an FTC-3000 qRT-PCR thermocycler (Funglyn Biotech, Toronto, Canada), applying the same procedure described in a previous study ( Tang et al., 2021 Tang et al., 2021 ). For the assessment of IGF2BP2 gene ( IGF2BP2 -Q-F: CCTGGGGGCTCTTCTCAG; IGF2BP2 -Q-R: GGATGTCTACCCGGGACTG) expression levels in goat ovarian tissue, we used glyceraldehyde-3-phosphate dehydrogenase ( GAPHD ) ( GAPDH- F: AAAGTGGACATCGTCGCCAT; GAPDH- R: CCGTTCTCTGCCTTGACTGT) as an internal control. 2.7 Statistical analyses and prediction of potential transcription factor-binding sites 2 Allelic and genotypic frequencies of the goat IGF2BP2 gene were analyzed in terms of Hardy-Weinberg equilibrium (HWE) using the Chi-square ( χ 2 ) test. We calculated genetic diversity indices using a web-based tool ( http://www.msrcall.com/Gdicall.aspx http://www.msrcall.com/Gdicall.aspx ) ( Jia et al., 2015 Jia et al., 2015 ). A polymorphism information content (PIC) of > 0.50 indicates a high level of polymorphism, whereas 0.25 < PIC≤0.50 and PIC≤0.25 represent moderate and low levels of polymorphism, respectively. In addition, we performed linkage disequilibrium (LD) analysis when two different loci were identified within the same gene, using the SHEsis platform ( http://analysis.bio-x.cn http://analysis.bio-x.cn ) ( Yong and He, 2005 Yong and He, 2005 ). We used a mixed-method linear model to determine the association between two indel mutations and goat litter size among the 885 SBWC goats with litter size information. The least-square means test was used to determine the correlation between different indel genotypes and litter size using the following formula: Y ij =‍ µ‍ + S i + G i + e ij , where Y ij represents the phenotypic value of each litter size; µ represents the overall average; S i represents the year of kidding; G i represents the fixed effect of the genotype; and e ij represents random error ( Wang et al., 2020 Wang et al., 2020 ). Using SPSS 20.0 software (IBM, USA), we analyzed the relationship between variants in the goat IGF2BP2 gene and litter size with analysis of variance (ANOVA) and the χ 2 test. All results were considered significant when P was < 0.05, and all statistical tests were performed as two-sided tests. In addition, we employed the JASPAR ( http://jaspar.genereg.net/collection/core http://jaspar.genereg.net/collection/core ) and Alibaba2 ( http://gene-regulation.com/pub/programs/alibaba2/index.html http://gene-regulation.com/pub/programs/alibaba2/index.html ) online databases to predict the combination of transcription factors (TFs) and mutation region sequences. 3 Results 1 3.1 Gene conservation analysis 2 NCBI BLAST analysis showed that the nucleotide sequence of C. hircus IGF2BP2 shared homology values with the sequences from O. aries , B. taurus , S. scrofa , H. sapiens , M. musculus , and G. gallus (99.44%, 99.22%, 94.10%, 91.26%, 85.05%, and 83.47%, respectively) ( Fig. 1 Fig. 1 ). In addition, phylogenetic tree analysis showed that the IGF2BP2 gene from C. hircus was most closely related to those from O. aries and B. taurus , whereas the relationship with G. gallus was the most distant ( Fig. 1 Fig. 1 ). 3.2 Association of expression of IGF2BP2 mRNA in ovaries with different fertility levels 2 In ovarian tissue obtained from adult female goats, the mRNA expression level of IGF2BP2 in the single-lamb group was significantly higher than that in the multi-lamb group ( Fig. 2 Fig. 2 ). 3.3 Indel selection and sequencing validation 2 After the DNA pooling test, we detected polymorphisms at two mutation sites among the six potential sites, for which primer pairs were designed ( Table 1 Table 1 ), including P4-Ins-13bp (13-bp insertion in IGF2BP2 ) and P5-Del-12bp (12-bp deletion in IGF2BP2 ). Both of these mutation sites in IGF2BP2 were associated with three potential genotypes, including homozygous insertion/insertion (II), heterozygous insertion/deletion (ID), and homozygous deletion/deletion (DD), which were identified by 3% agarose gel electrophoresis (Figs. 3a 3a and 3c 3c ). We selected two PCR products demonstrating the presence of indel mutations for Sanger sequencing, which identified two indels located in intron 2. The 12-bp deletion from the IGF2BP2 gene (12-bp indel NC_rs655441618: g. 81 116 754–81 116 765, Del AGGGTTTGCTGT) was consistent with the predicted indel information obtained from the NCBI database ( Fig. 3 Fig. 3 d). The 13-bp insertion in the IGF2BP2 gene (13-bp indel: g. ‍81 163 744‍–‍81 163 745, Ins TTTCCTTTTCTGG) was not completely consistent with that reported by the NCBI database ( Fig. 3 Fig. 3 b). 3.4 Genotypic and allelic frequencies and population indices 2 For the P4-Ins-13bp, the frequency of the wild-type genotype (DD) was higher than those for the II and ID genotypes ( Table 2 Table 2 ). Similarly, the frequency of the wild-type P5-Del-12bp genotype (II) was higher than those for the other genotypes. The two indel loci identified in IGF2BP2 did not conform to the HWE ( P < 0.05). According to the PIC values, the two detected IGF2BP2 mutations in SBWC goats were characterized as moderate polymorphisms (0.25 < PIC≤0.50) with PIC values ranging from 0.329 to 0.351. IGF2BP2 , insulin-like growth factor 2 mRNA-binding protein 2; SBWC, Shaanbei White Cashmere goat; II, homozygous insertion/insertion; ID, heterozygous insertion/deletion; DD, homozygous deletion/deletion; I, insertion; D, deletion; HWE, Hardy-Weinberg equilibrium; Ho, homozygosity; He, heterozygosity; Ne, effective allele number; PIC, polymorphism information content. 2 Locus 3 Genotypic frequency 2 Allelic frequency 2 HWE P value 4 Population parameter II ID DD D I Ho He Ne PIC P4-Ins-13bp 0.177 ( n =162) 0.345 ( n =317) 0.478 ( n =439) 0.651 0.349 <0.05 0.545 0.455 1.833 0.351 P5-Del-12bp 0.524 ( n =325) 0.363 ( n =223) 0.113 ( n =70) 0.295 0.705 <0.05 0.585 0.416 1.711 0.329 3.5 Linkage disequilibrium and haplotype analyses 2 To further explore whether the IGF2BP2 variant loci follow Mendelian inheritance patterns, we performed LD analysis using the SHEsis online platform. The D ' and r 2 values for the relationship between P5-Del-12bp and P4-Ins-13bp in the IGF2BP2 gene were 0.015 and 0.000, respectively, which indicated that these two mutations were not strongly linked ( Fig. 4 Fig. 4 ). The results of haplotype analysis for IGF2BP2 generated four haplotypes, and D P4-Ins-13bp -I P5-Del-12bp had the highest frequency ( Fig. 5 Fig. 5 ). 3.6 Association analysis between IGF2BP2 indels and litter size 2 To further evaluate the practical value of the detected mutations, the χ 2 test and correlation analyses were performed to assess the relationships between mutations and litter size. The χ 2 test results showed that the genotype ( χ 2 =14.479, P =0.006) distribution of the P4-Ins-13bp locus in the IGF2BP2 gene was significantly different between the single-lamb and multi-lamb groups ( Fig. 6 Fig. 6 a), whereas no significant differences were observed for the P5-Del-12bp locus ( Fig. 6 Fig. 6 c). At the same time, there was no significant difference in the distribution of I and D alleles at the two loci of IGF2BP2 between single-lamb and multi-lamb groups (Figs. 6b 6b and 6d 6d ), The association analysis showed that the P5-Del-12bp site of IGF2BP2 had no significant effect on litter size in SBWC goats. Importantly, at the P4-Ins-13bp site of IGF2BP2 , the litter size of individuals with the DD genotype was significantly higher than that of individuals with the ID and II genotypes ( P < 0.05; Table 3 Table 3 ). Therefore, for the P4-Ins-‍13bp indel in IGF2BP2 , the DD genotype was the favorable genotype. The data are expressed as mean±standard deviation. Values with different letters (a, b) within the same row differ significantly at P < 0.05 or P < 0.01. IGF2BP2 , insulin-like growth factor 2 mRNA-binding protein 2; SBWC, Shaanbei White Cashmere goat; II, homozygous insertion/insertion; ID, heterozygous insertion/deletion; DD, homozygous deletion/deletion; I, insertion; D, deletion. 2 Locus 3 Observed genotype 2 P value II ID DD P4-Ins-13bp 1.52±0.04 b ( n =159) 1.52±0.03 b ( n =305) 1.62±0.03 a ( n =421) 0.022 P5-Del-12bp 1.52±0.03 ( n =322) 1.57±0.06 ( n =217) 1.51±0.04 ( n =68) 0.348 3.7 Prediction of potential transcription-binding factors 2 The JASPAR online database predicted that the TFs, GATA-binding factor 2 (GATA2) [MA0036.3] and forkhead box L1 (FOXL1) [MA0033.2] , might bind to the P4-Ins-13bp and P5-Del-12bp insertion sequences in the IGF2BP2 , respectively ( Fig. 7 Fig. 7 ). 4 Discussion 1 Recent studies have identified IGF2BP2 as a candidate gene associated with litter size in goats, based on genome-wide selective sweep analysis (E et al., 2019). Additionally, IGF2BP2 expression has been detected in both resting and growing oocytes and granulocytes in mature mouse and human ovaries ( Hammer et al., 2005 Hammer et al., 2005 ). In this study, qRT-PCR analysis of adult female goat ovarian tissue indicated that the IGF2BP2 mRNA expression level in the single-lamb group was significantly higher than that in the multi-lamb group. This result indicates that IGF2BP2 might play a vital role in germ cells, embryonic development, and maturation, directly affecting fertility in goats. Thus far, most studies examining IGF2BP2 polymorphisms have focused on the effects of these polymorphisms on the risk of type 2 diabetes mellitus (T2DM), breast cancer, and lung cancer ( Zhao et al., 2012 Zhao et al., 2012 ; Liu et al., 2015 Liu et al., 2015 ; Rao et al., 2016 Rao et al., 2016 ; Chen et al., 2018 Chen et al., 2018 ). However, the association between IGF2BP2 indel polymorphisms and litter size in goats had not yet been examined; therefore, we explored the relationship in SBWC goats. In this study, two indel loci were detected in IGF2BP2 by sequencing, with PIC values ranging from 0.329 to 0.351, which indicated that these were moderate polymorphisms. However, the two indel loci in IGF2BP2 did not conform to the HWE ( P < 0.05), which might be due to genetic drift, artificial selection, or migration ( Wu et al., 2019 Wu et al., 2019 ). To further explore the relationship between the identified mutations and litter size, we performed the χ 2 test, which showed that the genotypic distributions of the IGF2BP2 P4-Ins-13bp mutation differed significantly between the single-lamb and multi-lamb groups. Correlation analysis showed that the 13-bp indel in IGF2BP2 was significantly associated with litter size in SBWC goats. Previous studies have reported that the absence of IGF2BP2 in mouse embryos resulted in the arrest of early embryonic development at the 2-cell-stage in vitro and the downregulation of cell division cycle and apoptosis regulator 1 ( Ccar1 ) and ribosomal protein S14 ( Rps14 ) expression, which are essential for early embryonic development ( Liu et al., 2019 Liu et al., 2019 ). Additionally, IGF2BP2 has been reported to promote the translation of IGF2 mRNA, and IGF2BP2 overexpression increases the level of IGF2 protein in cell lines ( Dai et al., 2011 Dai et al., 2011 ; Mu et al., 2015 Mu et al., 2015 ). IGF2 is a key regulator of embryo development and follicular maturation ( Hammer et al., 2005 Hammer et al., 2005 ), and polymorphisms in IGF2 exert significant impacts on the litter sizes of pigs of different parities ( Muñoz et al., 2010 Muñoz et al., 2010 ). These data suggest that IGF2BP2 may be involved in animal reproduction and that polymorphisms in this gene might influence the litter sizes of goats by influencing IGF2BP2 gene expression. However, the mechanism through which IGF2BP2 mutations alter goat reproduction requires further study. The two indels identified in this study were both located in intron 2 of IGF2BP2 , indicating that mutations in non-coding regions can affect mammalian reproductive traits. For instance, a 13-bp indel within the lysine methyltransferase 2A ( KMT2A ) gene and a novel 12-bp indel within the GDF9 gene are significantly associated with goat litter size ( Wang et al., 2017 Wang et al., 2017 ; Tang et al., 2021 Tang et al., 2021 ). The single-nucleotide polymorphism (SNP) mutation (c.2366G > A) in the 3' untranslated region (UTR) of bone morphogenetic protein 15 ( BMP15 ) has also been demonstrated to significantly affect the reproductive traits of Large White pigs ( Yin et al., 2019 Yin et al., 2019 ). To date, many studies have shown that non-coding regions can influence gene expression by altering the binding of TFs. For example, xeroderma pigmentosum group A (XPA)-binding protein 2 ( XAB2 ) gene deletion leads to the retention of RNA polymerase II subunit A ( POLR2A ) introns, weakening overall transcription and promoting cell senescence ( Hou et al., 2019 Hou et al., 2019 ). The bioinformatics analysis described in this study showed that the 13-bp indel sequence might alter GATA2 TF binding. GATA2 is essential for the differentiation of endometrial stromal cells (ESCs) ( Kin et al., 2015 Kin et al., 2015 ), and both GATA2 and GATA4 are involved in transcriptional regulation of gonadotropin genes ( Zheng et al., 2015 Zheng et al., 2015 ). In summary, IGF2BP2 may bind GATA2 through the P4-Ins-13bp insertion sequence, reducing the mRNA expression of IGF2BP2 in the multi-lamb group. 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Gonadotropin-releasing hormone and gonadal steroids regulate transcription factor mRNA expression in primary pituitary and immortalized gonadotrope cells . Reprod Sci , 22 ( 3 ): 285 - 299 . https://doi.org/10.1177/1933719114565031 https://doi.org/10.1177/1933719114565031 Gonadotropin-releasing hormone and gonadal steroids regulate transcription factor mRNA expression in primary pituitary and immortalized gonadotrope cells Xianyong LAN and Dongyun XIN designed the experiment; Haijing ZHU, Lei QU, and Libang HE provided goat samples and data records; Dongyun XIN and Yangyang BAI carried out all the experiments; Dongyun XIN prepared all figures and tables and drafted the manuscript; Yi BI, Yuxin KANG, Hong CHEN, Xianyong LAN, and Chuanying PAN checked and revised the manuscript. All authors have read and approved the final manuscript and, therefore, have full access to all the data in the study and take responsibility for the integrity and security of the data. This study was supported by the National Natural Science Foundation of China (Nos. 32060734 and 31760650). 10.1631/jzus.B2100079 1673-1581（2021）09-0757-10 2021-01-26 2021-03-31 2021-09-15 2022-05-09 2021-07-14 © Zhejiang University Press 2021 Dongyun XIN, Yangyang BAI, Yi BI, Libang HE, Yuxin KANG, Chuanying PAN, Haijing ZHU, Hong CHEN, Lei QU, and Xianyong LAN declare that they have no conflict of interest. The animal participants involved in this experiment were maintained in strict accordance with the “Regulations on the Management of Experimental Animal Affairs” (Ministry of Science and Technology of China, 2004) and this study was approved by the International Animal Care and Use Committee (IACUC) of Northwest A&F University, Yangling, China (Protocol No. NWAFAC1008). Compliance with ethics guidelines 浙江大学学报（英文版）（B辑：生物医学和生物技术） 9 22