Characterization and expression of DNA sequences encoding the growth hormone gene in African Pygmy Mouse (Mus minutoides)

Abstract

Keywords: body size, evolution, growth hormone, mouse, Mus minutoides

Mammals have various body sizes, ranging from large whales to small rodents. Each species
also has an inherent size, but it is not clear how this is determined and maintained.
Mus minutoides (African pygmy mouse) is one of the smallest mammals in the
world, measuring about 30 mm in body length and about 3 g in body weight. M.
minutoides originally inhabit the south of Saharan in Africa and is now bred as a
pet animal in many countries [5, 7]. The sexual maturity of M. minutoides is about 8 weeks
old, the gestation period is about 20 days, and the lifespan is about 2 years [15], which is similar to that of the common laboratory
mice, but the chromosomes of M. minutoides have unique characteristics that
differ from those of Mus musuclus (House mouse). There are individuals that
differ with different numbers of chromosomes in M. minutoides [7]. Moreover, M. minutodes has a special
sex-determination pattern [12]. However M.
minutoides is a quite small mammal, and despite its obvious characteristics, few
studies have focused on its size.

Many factors seem to be involved in determination of unique body size and growth rates in
different animal species, and they are thought to interact in complex manner. One of the most
representative factors involved in growth is growth hormone (Gh). Many studies have been
conducted to identify the structure, functional and binding sites of Gh and Gh receptors, and
to determine the regulatory mechanisms of growth and homeostasis [1, 4, 8, 10, 16]. In animals, Gh has been also well-studied, and the sequences of the
Gh gene have been identified not only in rodents but also in dogs [2], cats [6], and
pandas [11], and so on.

In general, dwarfism is also due to impaired function of growth hormone regulators in the
anterior pituitary gland and neuroendocrine and tissue transcription factors in the anterior
pituitary in mice [3, 14]. In addition, understanding the regulatory mechanisms of Gh and other pituitary
hormones is particularly important in body size determination mechanisms because many
spontaneous dwarf mice may be deficient in pituitary hormones.

The purpose of this study is to elucidate the basic mechanisms that determine the size of
M. minutoides by analyzing firstly the nucleotide sequences of
Gh, which is involved in animal body size.

All animal experiments were approved by the Experimental Animal Care and Use Committee of
Yamaguchi University (protocol number: 291). M. minutoides used in this study
were prepared for experiments immediately after purchase from different pet stores (Increase,
Inc., Himeji, Japan and Yanohashi Pet Trading, Yamaguchi, Japan). The species was determined
from appearance and body weight at 25 weeks old (Supplementary Fig. 1). M. musculus
(C57BL/6J) were purchased from Japan SLC (Hamamatsu, Japan) and housed in groups with
ad libitum access to water and food.

Primers were designed based on the nucleotide sequence of the Ensembl database (Mouse
(GRCm38.p6)) of the Gh gene, and the genomic DNA extracted from the tail
(n=3) was used for sequencing. The primers used for nucleotide sequencing were designed to
divide the entire gene length into three regions based on the sequence of M.
musculus. The primer sequences are listed in .

The Gh gene was amplified by PCR using the genome extracted from M.
minutoides and compared with M. musculus. The Gh
gene of M. musculus consists of a 1595 bp sequence and contains five exons.
Alignment of the nucleotide sequence of M. musculus showed substitutions,
deletions, and insertions in all regions including exons in M. minutoides
(). The DNA sequence of the Gh gene of M. minutoides
was registered to DNA Data Bank of Japan (accession number: LC5671320).

Alignment of a nucleic acid sequence of the genomic region and a putative amino acid
sequence of the Gh gene. (A) Alignment of a nucleic acid sequence of
the genomic region of the Gh gene between Mus musculus
and Mus minutoides. Arrows indicate primers for the nucleotide sequence
and qRT-PCR. The upper row is the sequence of M. musculus and the lower
row is that of M. minutoides. The region between right-angled arrows is
the genomic sequence of the Gh gene in M. musculus. *
indicates matching nucleic acids between M. musculus and M.
minutoides. Deletions and insertions across the entire region, including
exons, were observed in the genomic DNA of M. minutoides. The shaded
indicates five exons of M. musculus. (B) Alignment of a putative amino
acid sequence of Gh between M. musculus and M.
minutoides. The 25th and 37th amino acid sequences of M.
musculus are serine, respectively, while two sites are different in
M. minutoides: glycine and alanine.

Furthermore, the sequence of M. minutoides of the Gh gene
was translated into a putative amino acid sequence and compared with the amino acid sequence
of M. musculus using BLAST. The length of the putative amino acid sequence
was 216 amino acids in M. minutoides, and identical to that of M.
musculus (). At the DNA sequence
level, the Gh gene in M. minutoides differed from those in
M. musculus, but the homology at the putative amino acid level was very
high except for two amino acids, suggesting that M. minutoides also may have
a functional Gh. The results showed that only the 25th and 37th amino acid sequences of
M. musculus are serine, respectively, while two sites are different in
M. minutoides: glycine and alanine. These mutations were found in the
N-terminal region responsible for signal transduction and in the region that promotes Gh
protein maturation, respectively [9]. The 25th amino
acid in M. musculus is serine, while the 25th amino acid in M.
minutoides is glycine, suggesting that the binding ability of Gh protein to
receptors may be altered (www.uniprot.org/uniprot/{“type”:”entrez-protein”,”attrs”:{“text”:”P06880″,”term_id”:”134708″,”term_text”:”P06880″}}P06880/protvista). Serine, the 37th
amino acid in M. musculus, is part of the site responsible for the maturation
of the Gh protein, suggesting that the maturation process or post-translational modification
of the Gh protein in M. minutoides may be different from that in M.
musculus. M. minutoides purchased from the pet store was
maintained in closed colonies. Since the sequences were identical in M.
minutoides purchased from different pet stores, the sequences we found in this
study are considered to be unique to M. minutoides.

Next, to elucidate the evolutionary characteristics of the amino acid sequence of Gh in
M. minutoides, we constructed a phylogenetic tree of Gh in other
Mus genus and mammals () by the neighbor-joining method using the Poisson model by MEGAX with 1,000 bootstraps
(The Biodesign Institute, Tampe, AZ, USA), suggesting that the Gh of M.
minutoides diverged earlier than that of M. musculus but evolved
within the Mus genus. Therefore, we considered that not only the sequence of
the Gh but also its regulatory mechanism may be different between M.
minutoides and M. musculus.

Phylogenetic tree of the vertebrate Gh. The tree was constructed with the
neighbor-joining method using MEGAX from Mus minutoides and 13 other
vertebrate Gh amino acid sequences. Gh amino acid sequences were obtained from the
Ensembl database. Numbers on nodes represent the frequency with which the node is
recovered per 100 bootstrap replications in a total of 1,000.

Finally, we analyzed the Gh gene expression using qRT-PCR in the pituitary
gland of adult male M. minutoides (n=3). lists the primers used to detect Glyceraldehyde 3-phosphate dehydrogenase
(Gapdh) and Gh. We found that the expression of the
Gh gene was higher in M. minutoides than in M.
musculus (). It is unlikely that the sequence of the Gh gene is directly related
to the secretion of Gh protein, but the relatively high expression of the Gh
gene in M. minutoides may be related to body size, such as in association
with other growth-related factors. In dogs, growth promotion has been correlated with the
level of Igf1, a downstream factor of Gh, rather than with the Gh secretory [9]. In our preliminary experiments, we have found that the
expression level of both the Igf1 gene and Growth hormone
receptor (Ghr) gene in M. minutoides is much
lower than that in M. musculus. Igf1 is mainly secreted from the liver, which
is triggered by Gh signaling [13]. Although further
studies on the regulation of Gh gene expression in M.
minutoides are needed, it is possible that Gh gene expression may
be promoted by a decrease in Ghr gene expression and subsequent decrease in
Igf1 gene expression and blood levels.

Transcript levels of Gh in the pituitary gland of Mus
minutoides and Mus musculus. Left: The relative expression
level for Gh was normalized against the Gapdh
expression level. The expression of the Gh gene in M.
minutoides was higher than those in M. musculus. Data
represent the mean ± standard deviation. Right: RT-PCR analysis was performed for
confirmation of the specificity of the primer set.

The present study suggests that Gh of M. minutoides evolved within the
Mus genus and may have different functions and signaling from M.
musculus at the presumed amino acid sequence level. Furthermore, the expression
analysis of the Gh indicated that the regulatory mechanism of the
Gh in M. minutoides may differ from that in M.
musculus. In order to further elucidate the characteristic growth-related
characteristics of M. minutoides, particularly Gh, it will be necessary to
analyze related factors such as growth hormone receptors and Igf1.