Transcriptomic Analysis Reveals Insights on Male Infertility in Octopus maya Under Chronic Thermal Stress

Abstract

Octopus maya endemic to the Yucatan Peninsula, Mexico, is an ectotherm organism particularly temperature-sensitive. Studies in O. maya females show that temperatures above 27°C reduce the number of eggs per spawn, fertilization rate and the viability of embryos. High temperatures also reduce the male reproductive performance and success. However, the molecular mechanisms are still unknown. The transcriptomic profiles of testes from thermally stressed (30°C) and not stressed (24°C) adult male octopuses were compared, before and after mating to understand the molecular bases involved in the low reproductive performance at high temperature. The testis paired-end cDNA libraries were sequenced using the Illumina MiSeq platform. Then, the transcriptome was assembled de novo using Trinity software. A total of 53,214,611 high-quality paired reads were used to reconstruct 85,249 transcripts and 77,661 unigenes with an N50 of 889 bp length. Later, 13,154 transcripts were annotated implementing Blastx searches in the UniProt database. Differential expression analysis revealed 1,881 transcripts with significant difference among treatments. Functional annotation and pathway mapping of differential expressed transcripts revealed significant enrichment for biological processes involved in spermatogenesis, gamete generation, germ cell development, spermatid development and differentiation, response to stress, inflammatory response and apoptosis. Remarkably, the transcripts encoding genes such as ZMYND15, KLHL10, TDRD1, TSSK2 and DNAJB13, which are linked to male infertility in other species, were differentially expressed among the treatments. The expression levels of these key genes, involved in sperm motility and spermatogenesis were validated by quantitative real-time PCR. The results suggest that the reduction in male fertility at high temperature can be related to alterations in spermatozoa development and motility.

Keywords: RNA-Seq; chronic thermal stress; inflammation; male infertility; reproduction.

FIGURE 1

(A) Venn diagram of the number of transcripts expressed in the reference testis transcriptome of Octopus maya in each treatment. (B) Venn diagram of DETs with significant higher expression in each treatment. Treatments: 24PRE – control treatment exposed to 24°C; 24POST – mated and exposed to 24°C; 30PRE – exposed to 30°C; 30POST – mated and exposed to 30°C.

FIGURE 2

(A) E-value distribution of the Blastx hits against the UniProt and Non-redundant (Nr) database for each transcript. (B) Similarity distribution of the Blastx hits against the UniProt and Nr database. (C) Species distribution of the top blast hits of the transcripts in the Nr database in the testis transcriptomic analysis of O. maya males. (D) Species distribution of the top blast hits of the transcripts in the UniProt database in the testis transcriptomic analysis of O. maya males.

FIGURE 3

Gene ontology (GO) distribution by category at level 3 in the reference testis transcriptome of O. maya males.

FIGURE 4

Heatmap of the abundance of differentially expressed transcripts (rows, FDR < 0.05, Fold change > 2) in the O. maya testis transcriptome in each treatment (columns). The dendrogram shows that temperature modulated the expression patterns. Treatments: 24PRE – control treatment exposed to 24°C; 24POST – mated and exposed to 24°C; 30PRE – exposed to 30°C; 30POST – mated and exposed to 30°C.

FIGURE 5

Enriched GO terms of biological processes (Fisher exact test, FDR < 0.05) in 24POST treatment vs. the control treatment (24PRE). Up (red) and down-regulated (blue) transcripts are shown.

FIGURE 6

Enriched GO terms of biological processes (Fisher exact test, FDR < 0.05) in 30PRE treatment vs. the control treatment (24PRE). Up (red) and down-regulated (blue) transcripts are shown.

FIGURE 7

Enriched GO terms of biological processes (Fisher exact test, FDR < 0.05) in 30POST treatment vs. the control treatment (24PRE). Up (red) and down-regulated (blue) transcripts are shown.

FIGURE 8

Heatmap representing the expression values of differentially expressed transcripts (FDR < 0.05, Fold change > 2) between 24PRE, 24POST, 30PRE, and 30POST treatments and their main function. (A) Transcripts involved in the stress response. (B) Transcripts involved in reproductive process. Sample names are represented in columns and significant transcripts are represented in rows. Transcripts are clustered together based on expression similarity. Low to high expression is represented by a change of color from purple to yellow, respectively.

FIGURE 9

Relative expression of transcripts associated with thermal stress response. Mean values (back-transformed from logarithms) are shown in bars and letters are used to denote differences between means (P < 0.05).

FIGURE 10

Relative expression of transcripts associated with male reproductive process. Mean values (back-transformed from logarithms) are shown in bars and letters are used to denote differences between means (P < 0.05).