Heat stress induced piRNA alterations in pachytene spermatocytes and round spermatids

Abstract

Background: Spermatogenesis is a temperature-sensitive process, and elevation in temperature hampers this process quickly and significantly. We studied the molecular effects of testicular heating on piRNAs and gene expression in rat testicular germ cells.

Methods: We generated a cryptorchid rat model by displacing the testis from the scrotal sac (34 °C) to the abdominal area (37 °C) and sacrificed animals after 1 day, 3 days, and 5 days. Pachytene spermatocytes and round spermatids were purified using elutriation centrifugation and percoll gradient methods. We performed transcriptome sequencing in pachytene spermatocytes and round spermatids to identify differentially expressed piRNAs and their probable targets, i.e., TE transcripts and mRNAs.

Results: As a result of heat stress, we observed significant upregulation of piRNAs and TE transcripts in testicular germ cells. In addition to this, piRNA biogenesis machinery and heat shock proteins (Hsp70 and Hsp90 family members) were upregulated. mRNAs have also been proposed as targets for piRNAs; therefore, we shortlisted certain piRNA-mRNA pairs with an inverse relationship of expression. We observed that in testicular heat stress, the heat shock proteins go hand-in-hand with the upregulation of piRNA biogenesis machinery. The dysregulation of piRNAs in heat-stressed germ cells, increased ping-pong activity, and disturbed expression of piRNA target transcripts suggest a connection between piRNAs, mRNAs, and TE transcripts.

Conclusions: In heat stress, piRNAs, piRNA machinery, and heat shock proteins are activated to deal with low levels of stress, which is followed by a rescue approach in prolonged stressaccompained by high TE activity to allow genetic mutations, perhaps for survival and adaptability.

Keywords: Heat stress; Male infertility; Pachytene spermatocytes; Round spermatids; piRNA.

Fig. 1

Germ cell preparation: Cell fractions collected after centrifugal elutriation and acridine orange stained purified pachytene spermatocytes and round spermatids after centrifugal elutriation and percoll gradient

Fig. 2

piRNA classification: Classification of piRNAs into repeat-associated and non-repeat associated (A), the distribution of repeat-associated piRNAs into various repeat elements (B), the distribution of non ra-piRNAs into various genic features (C), the classification of all piRNAs into various genomic features (D)

Fig. 3

Characteristic features of piRNAs: Chromosomal distribution of all piRNAs in spermatocytes and spermatids (upper panel), percent nucleotide composition of all piRNA sequences as per the nucleotide position (lower panel)

Fig. 4

piRNA clusters predicted using proTRAC: piRNA clusters common in pachytene spermatocytes and round spermatids (A), Four biggest piRNA clusters in pachytene spermatocytes were present on chromosome 1 (B), chromosome 3 (C), chromosome 4 (D), and chromosome 20 (E)

Fig. 5

Differential expression of piRNAs in heat stress: heatmap visualization of differentially expressed piRNAs in spermatocytes (A) and spermatids (B). Venn diagram for commonly upregulated and down-regulated piRNAs (C-F)

Fig. 6

Ping-pong signature and TE expression analysis: Ping-pong signatures were identified in the small RNA read pairs in normal spermatocytes (A) 3DCR spermatocytes (B) and 5DCR spermatocytes (C) Normalized ping pong reads comparison in NC, 3DCR and 5DCR (D), TE transcripts differential expression in NC vs. 3DCR (E) NC vs. 5DCR (F), upregulated repeat families in 5DCR spermatocytes (G)

Fig. 7

Differential gene expression analysis of mRNAs in heat stress. The list of upregulated transcripts in 3DCR spermatocytes (A), upregulation of piRNA biogenesis players  (B), RT-PCR validation of the NGS data (C)

Fig. 8

piRNA targets downstream annotation: Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses for piRNA targets

Fig. 9

Representative piRNA-mRNA pairs showing inverse relation in transcriptome analysis. Upregulated piRNAs and their downregulated target expression in heat stressed spermatocytes (A). Downregulated piRNAs and their upregulated targets expression in heat stressed spermatocytes (B)