**2. The axis of hypothalamo-pituitary-thyroid**

Hypothalamus synthesizes and secrets thyrotropin-releasing-hormone (TRH) to send regulating information to pituitary (see Figure 1). TRH binds to the thyrotropin-releasinghormone receptors (TRHR) on the thyrotroph cells of pituitary to activate the intracellular signal pathways and induce the secretion and synthesis of thyrotropin (TSH). Circulating TSH in blood then binds to the thyrotropin receptors (TSHR) on the follicular cells of thyroid to activate the synthesis and secretion of thyroid hormone (TH).

TH secreted by thyroid is important to growth, development, and protein, fat, and carbohydrate metabolisms (Porterfield and White, 2007). It acts on almost all the organs and tissues. Each individual has a unique thyroid function set-point, and this set-point was suggested to be genetically determined (Hansen et al., 2004). Genetic variations of the hormones of HPT axis and their respective receptors could be the excellent candidates as the causing of related phenotype variations.

#### **2.1 Thyrotropin releasing hormone gene (***TRH***)**

Thyrotropin releasing hormone, produced in the paraventricular nucleus of the hypothalamus, is fully conserved in all species from human to bony fish that have been

Molecular Characterization of Hypothalamo-Pituitary-Thyroid Genes in Pig (Sus Scrofa) 547

Fig. 2. The schematic map of the porcine *TRH* gene. Boxes represented the exons (according to Wallis), of which the regions filled with diagonal lines were un-translated regions (UTR), the region filled with dots represented the signal peptide, and the regions full filled with

TRH initiates its effects by interacting with its receptor TRHR in the anterior pituitary. TRHR is a seven transmembrane spanning receptor and belongs to the G protein-coupled receptor superfamily. Actually, to date in total of three TRH receptors have been reported, each encoded by their specific genes. The first subtype of TRHR found in 1990 have been described in many species, e.g. mouse, rat, human, cow, chicken, frog and fish (Sun et al., 2003). However, the second subtype of TRHR identified in 1998 has only been reported in rodents, frog and fish. Further information of human genome sequence does not support the existence of the second TRHR subtype (Pfleger et al., 2004). The third TRHR subtype has only been reported in frog and fish (Mekuhi et al., 2010) so far. Figure 3 shows the evolution

Fig. 3. Polygenetic tree of TRHR proteins, aligned with method Clustal W. The branch of fish

Porcine *TRHR* gene (the first subtype) has been cloned and characterized recently (Jiang et al., 2011). It contains an open reading frame encoding 398 amino acids and shares 96.2%

used the TRHR sequence of *Catostomus commerson*.

black color represented the six copies of TRH precursor peptides.

**2.2 Thyrotropin Releasing Hormone Receptor gene (***TRHR***)** 

tree of the TRHR proteins.

investigated so far (Harder, 2001). It is the tripeptide pyro-Glu-His-Pro-NH2 derived from the preprohormone gene *TRH*, which also produces other non-TRH active peptides e.g. ppTRH160-169(pST10) and ppTRH178-199 (pFE22).

Fig. 1. Schematic map of hypothalamo-pituitary-thyroid axis regulatory network, according to Porterfield and White (2007).

No experiments on the porcine *TRH* gene have been done previously. However genomic information of this gene has became available as a result of the porcine whole genome sequencing, and comprehensive sequence analysis on it with bioinformatics method has been done (Wallis, 2010). Porcine *TRH* gene was found to be 3, 136 bp with 3 exons and two introns. A conserved signal peptide of 24 amino acids was predicted to be present, and there existed six copies of TRH sequences in the preprohormone peptide.

investigated so far (Harder, 2001). It is the tripeptide pyro-Glu-His-Pro-NH2 derived from the preprohormone gene *TRH*, which also produces other non-TRH active peptides e.g.

Fig. 1. Schematic map of hypothalamo-pituitary-thyroid axis regulatory network, according

No experiments on the porcine *TRH* gene have been done previously. However genomic information of this gene has became available as a result of the porcine whole genome sequencing, and comprehensive sequence analysis on it with bioinformatics method has been done (Wallis, 2010). Porcine *TRH* gene was found to be 3, 136 bp with 3 exons and two introns. A conserved signal peptide of 24 amino acids was predicted to be present, and there

existed six copies of TRH sequences in the preprohormone peptide.

ppTRH160-169(pST10) and ppTRH178-199 (pFE22).

to Porterfield and White (2007).

Fig. 2. The schematic map of the porcine *TRH* gene. Boxes represented the exons (according to Wallis), of which the regions filled with diagonal lines were un-translated regions (UTR), the region filled with dots represented the signal peptide, and the regions full filled with black color represented the six copies of TRH precursor peptides.

#### **2.2 Thyrotropin Releasing Hormone Receptor gene (***TRHR***)**

TRH initiates its effects by interacting with its receptor TRHR in the anterior pituitary. TRHR is a seven transmembrane spanning receptor and belongs to the G protein-coupled receptor superfamily. Actually, to date in total of three TRH receptors have been reported, each encoded by their specific genes. The first subtype of TRHR found in 1990 have been described in many species, e.g. mouse, rat, human, cow, chicken, frog and fish (Sun et al., 2003). However, the second subtype of TRHR identified in 1998 has only been reported in rodents, frog and fish. Further information of human genome sequence does not support the existence of the second TRHR subtype (Pfleger et al., 2004). The third TRHR subtype has only been reported in frog and fish (Mekuhi et al., 2010) so far. Figure 3 shows the evolution tree of the TRHR proteins.

Fig. 3. Polygenetic tree of TRHR proteins, aligned with method Clustal W. The branch of fish used the TRHR sequence of *Catostomus commerson*.

Porcine *TRHR* gene (the first subtype) has been cloned and characterized recently (Jiang et al., 2011). It contains an open reading frame encoding 398 amino acids and shares 96.2%

Molecular Characterization of Hypothalamo-Pituitary-Thyroid Genes in Pig (Sus Scrofa) 549

Quantitative trait loci (QTLs) are regions of the chromosome that are found to be associated with particular phenotypic traits by statistical analysis. Thus far, the pig QTL database (Pig QTLdb) has collected 6,344 QTLs from 281 publications in the past more than ten years. Any genes in these regions might be the positional candidate genes underlying the respective traits. Mapping the genes of HPT axis would help us to evaluate the potential genetic effects

In April 2009, the International Swine Genome Sequencing Consortium has completed and released a 4 sequence depth draft (Sscrofa9) by a minimal tile path BAC by BAC approach. This assembly can be conveniently accessed by the web-based query on the Ensemble (http://www.ensembl.org/Sus\_scrofa/Info/Index). Though a more recent assembly Sscrofa 10, a mixed BAC and WGS-based assembly of the porcine genome, has been released in April 2011, assemble errors in it remain to be resolved. Furthermore, the Pig QTLdb offered GBrowse map view of QTLs (http://www.animalgenome.org/cgi-bin/gbrowse/pig/) based on the Sscrofa 9. Thus, with the chromosome position of the candidate gene obtained by querying the Ensemble, one can easily get the information of the QTLs which were

Fig. 4. Query result of pig QTLdb Gbrowse map view, using TRH as an example.

Porcine *TRH* gene is located between 57,837,217 and 57,838,594 on chromosome 13. Based on the pig QTLdb, there are 20 QTLs including average daily gain (ADG), backfat weight, meat

**3. The gene mapping and expression analysis of these genes** 

of the variations of these genes.

mapped onto the same genomic region (Figure 4).

**3.1 The electrical mapping** 

amino acid identity to human TRHR. An intron disrupts the open reading frame in the sequence encoding the putative third intracellular loop which is between the fifth and sixth transmembrane domain. Besides, alternative spliced transcript variants and multiple transcription start sites have been observed in porcine *TRHR* gene (Jiang, 2011). The biological functions of these variants remain investigations.
