Characterizing Transcriptional Networks in Male Rainbow Darter (Etheostoma caeruleum) that Regulate Testis Development over a Complete Reproductive Cycle

• Published in: PloS one 2016-11, Vol.11 (11), p.e0164722-e0164722
• Main Authors: Bahamonde, Paulina A, McMaster, Mark E, Servos, Mark R, Martyniuk, Christopher J, Munkittrick, Kelly R
• Format: Article
• Language: English
• Subjects: Amino acids; Androgen receptors; Animals; Aromatase; Assembly; Bass; Biology; Biology and Life Sciences; Biosynthesis; Capacitation; Cell adhesion; Cell proliferation; Cluster Analysis; Computational Biology - methods; Developmental stages; Differences of sex development; Estrogen receptors; Estrogens; Etheostoma; Etheostoma blennioides; Etheostoma caeruleum; Fish; Fishes; Fishes - genetics; Fluidity; Gene expression; Gene Expression Profiling; Gene Ontology; Gene Regulatory Networks; Genes; Genetic aspects; Histology; Immune system; Male; Males; Medicine and Health Sciences; Membrane fluidity; Mesenchyme; Micropterus salmoides; Misgurnus anguillicaudatus; Morphology; Nitric oxide; Nitrogen oxides; Ontario; Pimephales promelas; Principal Component Analysis; Receptors; Regression analysis; Reproduction - genetics; Reproductive cycle; Research and Analysis Methods; Rivers; RNA; Seasons; Sewage; Sewage effluents; Sex differentiation; Signal transduction; Signaling; Spawning; Sperm; Spermatogenesis; Spermatozoa; Stem cells; Testes; Testis - metabolism; Transcription; Transcription (Genetics); Transcription, Genetic; Transportation networks; Zebrafish
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0164722

Intersex is a condition that has been associated with exposure to sewage effluents in male rainbow darter (Etheostoma caeruleum). To better understand changes in the transcriptome that are associated with intersex, we characterized annual changes in the testis transcriptome in wild, unexposed fish. Rainbow darter males were collected from the Grand River (Ontario, Canada) in May (spawning), August (post-spawning), October (recrudescence), January (developing) and March (pre-spawning). Histology was used to determine the proportion of spermatogenic cell types that were present during each period of testicular maturation. Regression analysis determined that the proportion of spermatozoa versus spermatocytes in all stages of development (R2 ≥ 0.58) were inversely related; however this was not the case when males were in the post-spawning period. Gene networks that were specific to the transition from developing to pre-spawning stages included nitric oxide biosynthesis, response to wounding, sperm cell function, and stem cell maintenance. The pre-spawning to spawning transition included gene networks related to amino acid import, glycogenesis, Sertoli cell proliferation, sperm capacitation, and sperm motility. The spawning to post-spawning transition included unique gene networks associated with chromosome condensation, ribosome biogenesis and assembly, and mitotic spindle assembly. Lastly, the transition from post-spawning to recrudescence included gene networks associated with egg activation, epithelial to mesenchymal transition, membrane fluidity, and sperm cell adhesion. Noteworthy was that there were a significant number of gene networks related to immune system function that were differentially expressed throughout reproduction, suggesting that immune network signalling has a prominent role in the male testis. Transcripts in the testis of post-spawning individuals showed patterns of expression that were most different for the majority of transcripts investigated when compared to the other stages. Interestingly, many transcripts associated with female sex differentiation (i.e. esr1, sox9, cdca8 and survivin) were significantly higher in the testis during the post-spawning season compared to other testis stages. At post-spawning, there were higher levels of estrogen and androgen receptors (esr1, esr2, ar) in the testis, while there was a decrease in the levels of sperm associated antigen 1 (spag1) and spermatogenesis associated 4 (spata4) mRNA. Cyp17a was