<i>De novo</i> sequencing and comparative analysis of testicular transcriptome from different reproductive phases in freshwater spotted snakehead <i>Channa punctatus</i>

<div><p>The spotted snakehead <i>Channa punctatus</i> is a seasonally breeding teleost widely distributed in the Indian subcontinent and economically important due to high nutritional value. The declining population of <i>C</i>. <i>punctatus</i> prompted us to focus on genetic regulation of its reproduction. The present study carried out <i>de novo</i> testicular transcriptome sequencing during the four reproductive phases and correlated differential expression of transcripts with various testicular events in <i>C</i>. <i>punctatus</i>. The Illumina paired-end sequencing of testicular transcriptome from resting, preparatory, spawning and postspawning phases generated 41.94, 47.51, 61.81 and 44.45 million reads, and 105526, 105169, 122964 and 106544 transcripts, respectively. Transcripts annotated using <i>Rattus norvegicus</i> reference protein sequences and classified under various subcategories of biological process, molecular function and cellular component showed that the majority of the subcategories had highest number of transcripts during spawning phase. In addition, analysis of transcripts exhibiting differential expression during the four phases revealed an appreciable increase in upregulated transcripts of biological processes such as cell proliferation and differentiation, cytoskeleton organization, response to vitamin A, transcription and translation, regulation of angiogenesis and response to hypoxia during spermatogenically active phases. The study also identified significant differential expression of transcripts relevant to spermatogenesis (<i>mgat3</i>, <i>nqo1</i>, <i>hes2</i>, <i>rgs4</i>, <i>cxcl2</i>, <i>alcam</i>, <i>agmat</i>), steroidogenesis (<i>star</i>, <i>tkt</i>, <i>gipc3</i>), cell proliferation (<i>eef1a2</i>, <i>btg3</i>, <i>pif1</i>, <i>myo16</i>, <i>grik3</i>, <i>trim39</i>, <i>plbd1</i>), cytoskeletal organization (<i>espn</i>, <i>wipf3</i>, <i>cd276</i>), sperm development (<i>klhl10</i>, <i>mast1</i>, <i>hspa1a</i>, <i>slc6a1</i>, <i>ros1</i>, <i>foxj1</i>, <i>hipk1</i>), and sperm transport and motility (<i>hint1</i>, <i>muc13</i>). Analysis of functional annotation and differential expression of testicular transcripts depending on reproductive phases of <i>C</i>. <i>punctatus</i> helped in developing a comprehensive understanding on genetic regulation of spermatogenic and steroidogenic events in seasonally breeding teleosts. Our findings provide the basis for future investigation on the precise role of testicular genes in regulation of seasonal reproduction in male teleosts.</p></div

Enlisting the selected genes and their primers for quantitative PCR.

Enlisting the selected genes and their primers for quantitative PCR.</p

Showing expression fold change of some differentially expressed testicular genes <i>tkt</i> (A), <i>mgat3</i> (B), <i>alcam</i> (C), <i>gipc3</i> (D) and <i>stx1b</i> (E) along the reproductive cycle.

<p>The expression fold change of genes during preparatory, spawning and postspawning phases were calculated using expression values obtained during resting phase as reference. Ribosomal 18s RNA was used as the house-keeping gene for normalization of expression values. Three testicular samples were used for each reproductive phase (N = 3). Data represented as mean ± SEM were analyzed by one way analysis of variance (ANOVA) and compared by Newman-Keuls multiple range test. Groups with different alphabets (a-b) as superscripts show significant difference (<i>P</i> < 0.05).</p

Histogram representation of gene ontology classification of transcripts from different reproductive phases.

<p>GO classification of testicular transcripts from different reproductive phases (resting: R, preparatory: P, spawning: S and postspawning: Ps) into various subcategories under Biological process (A), Molecular function (B) and Cellular component (C).</p

Venn diagram of testicular transcripts showing significant differential expression depending on reproductive phases (corrected <i>P</i> value < 0.05).

<p>Venn diagram of testicular transcripts showing significant differential expression depending on reproductive phases (corrected <i>P</i> value < 0.05).</p

Histogram representation of gene ontology classification of upregulated testicular transcripts from different reproductive phases.

<p>Classification of upregulated testicular transcripts from different reproductive phases (resting: R, preparatory: P, spawning: S and postspawning: Ps) associated with testicular functions under GO subcategories of Biological process (A), Cellular component (B) and Molecular function (C). Upregulated transcripts were obtained from each set of clustered transcripts (set 1: samples <u>R</u> and P; set 2: samples <u>P</u> and S; set 3: samples <u>S</u> and Ps; set 4: samples <u>Ps</u> and R) based on expression fold change that was calculated considering the underlined sample in each set as reference.</p

Heat map representation of significantly differentially expressed testicular transcripts.

<p>Shows testicular transcripts showing significant (corrected <i>P</i> value < 0.05) differential expression based on comparison between different reproductive phases (resting: R, preparatory: P, spawning: S and postspawning: Ps): (A) R and P, (B) P and S, (C) S and Ps and (D) R and Ps. Transcripts differentially expressed in more than two reproductive phases are labelled.</p