The gonadotropin-releasing hormone gene : characterization, regulation and expression in two salmonids
Date
2017-10-16
Authors
Von Schalburg, Kristian Robert
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Abstract
There are currently thirteen members of the gonadotropin-releasing
hormone (GnRH) family. The GnRH members that activate the synthesis and
release of the pituitary gonadotropins are the best understood. These
members stand central to the development and maintenance of
reproductive function. The roles of GnRH that act in the brain and not
in the pituitary, or that are expressed in extraneural tissues, are
not well characterized.
My goal was to determine whether 1) the regulatory regions and
organization of the GnRH gene is conserved between mammals and fish,
2) GnRH is expressed in tissues other than the brain of salmonids and
3) the processes that regulate the expression of GnRH are conserved
between two salmonid species with different habitats and reproductive patterns (sockeye salmon, Oncorynchus nerka; rainbow trout, Oncorynchus mykiss).
To determine whether the regulatory regions and organization of the
GnRH gene were conserved across the species, I isolated and
characterized salmon (s)GnRH gene 1 from rainbow trout and sGnRH gene2
from sockeye salmon. In salmon, which are tetraploid, each duplicated
sGnRH gene encoded a different mRNA (mRNA1 or mRNA2), but the
identical sGnRH peptide. A Southern blot analysis revealed that other
related forms of GnRH exist in the sockeye salmon genome. Also, I
determined from RT-PCR analysis that GnRH was not expressed in the
heart, liver, gut, adrenal, spleen or retina, but was expressed in the
gonads of sockeye salmon.
To understand the function of GnRH in the gonads, it was necessary to
learn when GnRH was expressed during development and throughout the
reproductive cycle. Studies using RT-PCR analysis and primer extension
analysis demonstrated that the reproductive tissues of salmonids use
an upstream promoter to regulate GnRH expression. Intron 1 may
be retained, resulting in mRNAs containing 5'-untranslated regions
longer than their brain counterparts. These sGnRH transcripts are
initiated by a TATA-less promoter region from a start site at 315
basepairs upstream from that utilized in the sockeye salmon brain.
Using the same techniques, differences in the expression of GnRH in
embryonic tissue and gonads of sockeye salmon and rainbow trout were
noted over the first two years of their lives. First, the upstream
promoter is transiently used for expression of GnRH as early as 14
days after fertilization in rainbow trout and 30 days after
fertilization in sockeye salmon. Second, in sockeye salmon ovary and
testis, GnRH was expressed in October of the first year and then only
during May and June of the second year in precocious tissue.
For rainbow trout, GnRH was expressed in the first year from May to
October and in the second year only in December. Precociously mature
ovary and testis expressed GnRH from June to October in the second
year.
It was also important to determine whether the GnRH mRNA expressed in
the developing ovary and testis was translated into protein. High
pressure liquid chromatography and radioimmunoassays were used to
demonstrate the presence of at least three forms of GnRH in
precociously mature ovaries and testes during the second year.
The expression of sGnRH mRNA2 in the salmonid ovary and testis
utilizes an alternative promoter. The resulting mRNAs have long 5'-
untranslated regions that may be important in post-transcriptional
control. Expression of GnRH in the brain is constant, but
is intermittant in the salmonid gonad. GnRH mRNA is expressed in
undifferentiated gonadal tissue in the first year and briefly in
differentiated, but immature gonads. However, in precociously mature
ovary and testis in the second year GnRH is transcribed and translated
at the stage that precedes ovulation and spawning. Differences in
pattern and longer duration of GnRH expression are shown in the ovary
and testis of rainbow trout in comparison to sockeye salmon. This
might indicate that GnRH is important in the regeneration of new sets
of germ cells in the iteroparous rainbow trout, but not in the
semelparous sockeye salmon.
A comparison of the genes that encode sGnRH mRNA1 and mRNA2 reveals
significant sequence divergence in their 5’-flanking regions following
tetraploidization. A large portion of the sockeye salmon gene2 is
missing in comparison to the Atlantic salmon gene2. However, the
salmonid genes all share strong sequence identity in the proximalprom
oter region. Although large segments of sequence identity do not exist
in the regulatory regions of the GnRH-encoding genes of mammals and
salmonids, some similarities exist in the positions of potential POU-
homeodomain regulator and estrogen response element motifs. This
suggests that some regulatory control for expression of GnRH in both
the brain and gonads may be conserved.
Description
Keywords
Salmonidae, Hormones