The gonadotropin-releasing hormone gene : characterization, regulation and expression in two salmonids

dc.contributor.authorVon Schalburg, Kristian Robert
dc.contributor.supervisorSherwood, Nancy of Biologyen_US of Philosophy Ph.D.en_US
dc.description.abstractThere 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.en_US
dc.rightsAvailable to the World Wide Weben_US
dc.titleThe gonadotropin-releasing hormone gene : characterization, regulation and expression in two salmonidsen_US


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