Molecular characterization and expression of TGFβRI and TGFβRII and its association with litter size in Tibetan sheep

Abstract Backgrounds Transforming growth factor‐β (TGF‐β) type I receptor (TGFβRI) and type II receptor (TGFβRII) are the members of the TGFβ superfamily, which are potent regulators of cell proliferation and differentiation in many organ systems, and they play key roles in multiple aspects of follicle development. Objectives We aimed to explore the characterization, expression analysis of TGFβRI and TGFβRII genes, and the association with litter size in Tibetan sheep. Methods In this study, we cloned the complete coding sequences of TGFβRI and TGFβRII genes in Tibetan sheep and analyzed their genomic structures. Results The results showed that percentages of sequences homology of the two proteins in Tibetan sheep were the most similar to Ovis aries (100%), followed by Bos mutus (99%). The RT‐qPCR showed that two genes were expressed widely in the different tissues of Tibetan sheep. The TGFβRI expression was the highest in the lung (p < 0.05), followed by the spleen and ovary (p < 0.05). The TGFβRII expression was significantly higher in uterus than that in lung and ovary (p < 0.05). In addition, the χ 2 test indicated that all ewes in the population were in Hardy–Weinberg equilibrium, and the population was in medium or low polymorphic information content status. We also found four Single Nucleotide Polymorphism (SNPs), g.9414A > G, g.28881A > G, g.28809T > C, g.10429G > A in sheep TGFβRI gene and g.63940C > T, g.63976C > T, g.64538C > T, g.64504T > A in TGFβRII gene. Three genotypes, except for g.64504T > A, and three haplotypes were identified in each gene. linkage disequilibrium analysis indicated that there was strong linkage disequilibrium in each gene. The association analysis showed that the four SNPs of TGFβRI were associated with litter size (p < 0.05), and g.63940C > T of TGFβRII was confirmed to be associated with litter size (p < 0.05). Conclusions Based on these preliminary results, we can assume that TGFβ receptors (TGFβRI and TGFβRII) may play an important role in sheep reproduction.


INTRODUCTION
The TGFβ superfamily is a large and expanding group of regulatory polypeptides (Kumari et al., 2021). The molecular signalling pathway of the TGFβ superfamily has been conserved throughout the six hundred million years of metazoan evolution (Loveland & Hime, 2005), which is critical for regulating a variety of developmental events, including cell proliferation, differentiation, and matrix secretion (Elvin et al., 2000;Nong et al., 2019). The family members of the TGFβ superfamily are candidates for mediating important oocyte activity (Elvin et al., 2000;Lankford & Weber, 2010). TGFβ receptor type I (TGFβRI) and the TGFβ receptor typeII (TGFβRII) are important members of the TGFβ superfamily. TGFβ signalling, important in ovary development is mediated through TGFβRI and TGFβRII. These receptors are interdependent components of a heteromeric complex, as receptor I requires receptor II for TGFβ binding and receptor II requires receptor I for signalling (Attisano & Wrana, 2002;Knight & Glister, 2003;Sun et al., 2008).
TGFβ ligands bind and activate TGFβ receptor complex composed of the type II (TGFβRII) and type I subunits (TGFβRI), which phosphorylate Smad2 and Smad3. Activated Smad2/3 forms transcriptional complexes with Smad4 and other transcriptional factors and regulates the transcription of genes (Serizawa et al., 2013). It has been reported that they play an important role in many aspects of follicular development, including activation of resting primordial follicles, proliferation and apoptosis of Granulosa cells and membrane cells, steroid formation, gonadotropin receptor expression, oocyte maturation, ovulation, and luteinization (Elvin et al., 2000). The various type I and type II receptors through which each of these ligands can signal are expressed by pre-granulosa cells/granulosa cells of the corresponding early follicle stages, making these cells potential targets for paracrine signalling (Shimasaki et al., 2004).
A few genes of the TGFβ superfamily were investigated, and their association with reproductive performance has been observed in lines of sheep (Elvin et al., 2000;Jia et al., 2020;Shi et al., 2021;Shimasaki et al., 2004;Xu et al., 2010

Tissue expression analysis of sheep TGFβRI and TGFβRII
The primers for real-time PCR were designed according to mRNA sequences of TGFβRI and TGFβRII gene (GenBank accession No: XM_004004226.4 and XM_012099309.2) ( Table 1). The reaction TA B L E 1 Primer information and PCR conditions used in this study

SNP identification and genotyping
TGFβRI and TGFβRII genes Single Nucleotide Polymorphism (SNPs) were screened Using the dbSNP database (http://www.ncbi.nlm.nih. gov/snp) and verified by DNA sequencing. Improved multiplex ligation detection reaction (iMLDR TM ) was used for genotyping following the instrument operating guidelines. Genotypic, allelic frequencies, and genetic parameters were directly calculated following previous description (Zhao et al., 2013). The linkage disequilibrium (LD) was conducted using the Haploview software.

Association analysis
The association analysis between genotypes and litter size of ewes was determined according to a general linear model (GLM) program. All statistical analyses were performed using SPSS 23.0. Results with p < 0.05 were considered significantly different. Based on the characteristics of sheep, the statistical model was as follows: where y ijn is the phenotypic value, μ is the population mean, Pi is the fixed effect of the ith parity (i = 1, 2, or 3), Gj is the fixed effect of the jth genotype (j = 1, 2, 3), I PG is the interaction effect of parity and genotype, and e ijn is the random residual.

Molecular cloning and sequence analysis of sheep TGFβRI and TGFβRII
In this study, 1751 bp of the sheep TGFβRI gene was cloned, which con-   Figures 1 and 2).
The structure prediction of sheep TGFβRI protein was performed by online protein analysis system SOPMA. The results showed that the extension chain composed of alpha-helix, extended strand, beta turn, and random coil accounted for 39.32%, 11.38%, 3.39%, and 67.27%, respectively, and for TGFβRII protein, they were 33.76%, 15.92%, 3.82% and 46.50%, respectively (Figures 3 and 4).

Expression profile analysis
The RT-qPCR was used to investigate the general tissue distributions of TGFβRI and TGFβRII. As shown in Figures 5 and 6, two genes were widely expressed in hypothalamus, pituitary, heart, liver, spleen, lung, kidney, ovary, oviduct, uterus, rumen, duodenum, and longissimus dorsi in Tibetan sheep. The TGFβRI was expressed with the highest level in the lung (p < 0.05), followed by the spleen, uterus and ovary (p < 0.05), and almost no expression in longissimus dorsi. The TGFβRII expression was the highest in uterus than in other tissues (p < 0.05), followed by lung, ovary, and spleen (p < 0.05). There were no significant differences among oviduct, duodenum, rumen, kidney, pituitary, liver, and heart (p > 0.05). Except for the hypothalamus, the expression of TGFβRII gene in longissimus dorsi was lower than that in the other tissues (p < 0.05).

Population genetic analysis of polymorphism in sheep TGFβRI and TGFβRII
In this study, four polymorphic nucleotide sites (SNPs) were identified in Tibetan sheep TGFβRI and TGFβRII genes, respectively. All mutations were synonymous mutations. Except for SNP g.64504T > A, the other SNPs were classified as three genotypes (Table 2), and three haplotypes were identified in each gene (Table 3). Linkage disequilibrium (r 2 ) block indicated strong linkage disequilibrium in two genes, respectively ( Figure 7). In addition, Ho, He, Ne, and polymorphic information content (PIC) of Tibetan sheep TGFβRI were 0.72, 0.28, 1.40, and 0.24, respectively, and for TGFβRII, 0.76, 0.24, 1.31, and 0.21, respectively. Tibetan sheep were in medium PIC status at g.63940C > T and g.28809T > C sites, and the others have low PIC status (Table 4). The χ 2 test indicated that all ewes in the populations were in Hardy-Weinberg equilibrium.

Association analysis of SNPs with litter size in Tibetan sheep
The effects of Tibetan sheep TGFβRI and TGFβRII SNPs on litter size of the experimental populations were studied. The results showed that the g.9414A > G, g.28881A > G, g.28809T > C, and g.10429G > A of sheep TGFβRI were associated with litter size (p < 0.05). In contrast, the TGFβRII g.63940C>T substitution was associated with litter size (p < 0.05). However, the SNPs, g.63976C > T, g.64538C > T, and g.64538C > T had no association with litter size (Table 5). All results indicated that TGFβRI and TGFβRII contributed to phenotype values.

DISCUSSION
TGFβ superfamily is evolutionarily conserved and plays fundamental roles in cell growth and differentiation (Attisano & Wrana, 1996;Hill, 1996). TGFβ superfamily signalling is essential for female reproduction (Li, 2014), and TGFβ superfamily affects the reproductive physiology of animals (Nie et al., 2014), for example, influencing the development of follicles by regulating the proliferation or apoptosis of Granulosa cells in the follicles and causing follicular atresia (Li, 2014;Nie et al., 2014;Ovchinnikov & Wolvetang, 2011 (Ester et al., 1999), so TGFβRI and TGFβRII genes were used as candidate genes for reproductive traits to study. TGFβRI and TGFβRII are serine-threonine kinases that signal through the Smad family of proteins (Ovchinnikov & Wolvetang, 2011;Sun et al., 2008). TGFβ1 binds to the TGFβRII, which in turn recruits the binding of TGFβRI to form a heterotetramer. TGFβRI then phosphorylates and activates the Smad2 protein (Li, 2014;Nie et al., 2014) after combining with Smad4, followed by translocation to the nucleus where the activated Smad complex. Then, it is involved in regulating transcriptional responses on target genes (Ikushima & Miyazono, 2010). At present, there are few studies on the structural characterization of TGFβRI and TGFβRII. In this study, we analyzed the homology of sheep TGFβRI and TGFβRII proteins with 10 other species, respectively. It was found that TGFβRI and TGFβRII have a higher percentage of sequences homology indicating that TGFβRI and TGFβRII were conserved across the above-mentioned species.
Type I and type II TGFβ receptors appear to be ubiquitously expressed in most cell types (Knight & Glister, 2006). The tissue to produce TGFβRI and TGFβRII, whose expression was first detected in preantral follicles and continues throughout the subsequent stages of follicular development (Knight & Glister, 2006). The mRNA and proteins of TGFβ receptors type I and II exist in the human oocyte, and receptor type I exists in blastocysts, indicating a selective expression of transcripts for TGFβ receptors in oocytes and blastocysts (Osterlund & Fried, 2000). Expression of TGFβRI mRNA was observed in the sheep ovary, while expression of TGFβRII mRNA within the follicle was limited to the theca (Juengel et al., 2004). The expression of TGFβR mRNA/protein in preantral follicles has been documented in several species including rodents, human, sheep, and cattle (Chow et al., 2001;Juengel et al., 2004;Osterlund & Fried, 2000;Roy, 2000). We found F I G U R E 3 Secondary structure of TGFβRI (a) and TGFβRII (b) protein. Blue represents alpha helix, green represents beta turn, purple represents random coil, and red represents extended strand. hypothalamus, and hypophysis, as well as in other tissues. We also found that expression of TGFβRI was the highest in lung, followed by spleen, uterus, and ovary, and TGFβRII was higher in uterus than in the other tissues.

TA B L E 3 Haplotypes of SNPs loci sites of TGFβR1 and TGFβRII
TGFβRI and TGFβRII are essential for regulating the growth and differentiation of ovarian follicles and thus fertility (Juengel et al., 2004). Osterlund and Fried (2000) reported that TGFβ receptor types I and II are present in human oocytes. Juengel et al. (2004) reported that the expression of mRNAs encoding TGF-β1 and TGF-β2 as well as both type I and II TGF-β receptors were observed in the theca of small growing follicles indicating that TGF-βs may be regulating thecal cell function in an autocrine manner. Expression of mRNA encoding TGF-β type I and II receptors was observed in luteal cells, stroma, the vascular system, and surface epithelium suggesting that TGF-βs may also regulate other cell types in the sheep ovary (Juengel et al., 2004). A similar pattern of expression for the TGFβRII mRNA was observed in mouse follicles, with expression most prominent in the theca and barely detectable in granulosa cells (Juengel et al., 2004 the association between the two genes and productive performance of different sheep breeds are required.

CONCLUSIONS
In this study, we cloned cDNA sequences of TGFβRI and TGFβRII genes in Tibetan sheep and the sequences homology of the two genes was the most similar to O. aries, followed by B. mutus. We also found that TGFβRI and TGFβRII were expressed in the different tissues of Tibetan sheep, and the expression of TGFβRI was the highest in lung, followed by spleen, uterus, and ovary, and TGFβRII expression was higher in uterus than the other tissues. The g.9414A > G, g.28881A > G, g.28809T > C, g.10429G > A mutations of TGFβRI and g.63940C > T of TGFβRII were screened out, and three different genotypes as well as three different haplotypes were identified for each gene. The g.9414A > G, g.28881A > G, g.28809T > C, and g.10429G > A mutation of sheep TGFβRI had an association with litter size, and the TGFβRII g.63940C > T was associated with litter size.