Transcriptome profiling of LH-producing gonadotropes in female medaka - with special emphasis on calcium-activated potassium channels

Abstract

The brain-pituitary-gonad axis regulates puberty and reproduction in all vertebrates. The two gonadotropins, FSH and LH are released from the pituitary and initiates maturing and growth of the gonads. To understand more of the changes that take place in the gonadotropes during puberty, the transcriptome of LH-producing gonadotropes from adult and juvenile medaka were profiled with high-throughput sequencing. The pituitaries were sampled from genotyped females from a (lhb:GFP) transgenic line. LH-producing cells were sorted out by FACS. RNA was isolated from the sorted cells, amplified, converted to cDNA, fragmented and sequenced with Illumina.

The sequence output from Illumina is called reads, and the reads were aligned to the medaka genome with software called TopHat. Reads were counted with the software HTSeq, and differentially expressed genes were located with the software DESeq. Some of the expressed genes that are known to be involved in reproduction, like, LHβ-subunit, GnRH-R’s and dopamine receptors were focused on. The LHβ-subunit was highly expressed in both adult and juveniles. The analysis shows low expression of GnRH-R-3 in the adults. The dopamine receptors D1, D2 and D3 were all expressed. Plenty of the differential expression analysis between juvenile and adult sample remains. However, the material has potential to contribute to the understanding how the gonadotropes mature.

In addition, KCa channel genes were investigated further since these channels are believed to be involved in the excitability of endocrine cells and thus important for secretion control. KCa channels were obtained from eel and cod, in addition to medaka by blasting their respective genomes with known KCa channel sequences. In eel only the BK channel, the SK1 channel and the SK2 channel sequences were obtained, while all the channel sequences were obtained from medaka and cod. The obtained sequences were analysed by sequence alignments and phylogenetic analysis, in addition to the output from RNA-seq. All KCa channels were expressed in both juvenile and female medaka, except for one variant of the SK1 channel. This particular variant of SK1 also showed a different phylogenetic relationship, than the other SK1 channels, suggesting that the channel type is non-functional. Phylogenetic analyses were performed on all the KCa channels to find the phylogenetic relationship according to species and channel type. The phylogenetic analyses showed the expected relationship according to channel type and species.