PHLDA1 Does Not Contribute Directly to Heat Shock-Induced Apoptosis of Spermatocytes.

Patryk Janus,Wiesława Widłak,Agnieszka Toma-Jonik,Natalia Vydra,Katarzyna Mrowiec,Joanna Korfanty,Piotr Widłak,Ryszard Smolarczyk

doi:10.3390/ijms21010267

Abstract

Spermatocytes are among the most heat-sensitive cells and the exposure of testes to heat shock results in their Heat Shock Factor 1 (HSF1)-mediated apoptosis. Several lines of evidence suggest that pleckstrin-homology-like domain family A, member 1 (PHLDA1) plays a role in promoting heat shock-induced cell death in spermatogenic cells, yet its precise physiological role is not well understood. Aiming to elucidate the hypothetical role of PHLDA1 in HSF1-mediated apoptosis of spermatogenic cells we characterized its expression in mouse testes during normal development and after heat shock. We stated that transcription of Phlda1 is upregulated by heat shock in many adult mouse organs including the testes. Analyzes of the Phlda1 expression during postnatal development indicate that it is expressed in pre-meiotic or somatic cells of the testis. It starts to be transcribed much earlier than spermatocytes are fully developed and its transcripts and protein products do not accumulate further in the later stages. Moreover, neither heat shock nor expression of constitutively active HSF1 results in the accumulation of PHLDA1 protein in meiotic and post-meiotic cells although both conditions induce massive apoptosis of spermatocytes. Furthermore, the overexpression of PHLDA1 in NIH3T3 cells leads to cell detachment, yet classical apoptosis is not observed. Therefore, our findings indicate that PHLDA1 cannot directly contribute to the heat-induced apoptosis of spermatocytes. Instead, PHLDA1 could hypothetically participate in death of spermatocytes indirectly via activation of changes in the somatic or pre-meiotic cells present in the testes.

Highlights

Elevated temperatures and other types of proteotoxic stresses lead to a Heat Shock Factor 1 (HSF1) activation, whose main targets are genes encoding molecular chaperones, primarily HSP proteins, providing cytoprotection [1]
The increased level of the PHLDA1 protein was detected by western blot in testes of mice subjected to heat shock (Figure 1b); it should be noted that the level of PHLDA1 was much lower in the testis than in the liver or NIH3T3 cells
We revealed that Phlda1 transcription begins in the early stages of mouse testicular development and its protein products do not accumulate in later developmental stages of spermatogenic cells

Summary

Introduction

Elevated temperatures and other types of proteotoxic stresses lead to a Heat Shock Factor 1 (HSF1) activation, whose main targets are genes encoding molecular chaperones, primarily HSP proteins, providing cytoprotection [1]. The expression of inducible Hsp genes is blocked in heat-shocked spermatocytes [2,3], while the expression of constitutively expressed testis-specific variants of HSP70 (HSPA2 and HSPA1L) is down-regulated after heat shock [4]. Spermatocytes and round spermatids are among the most heat-sensitive cells [7] and the most significant consequence of the heat stress in testes is the loss of germ cells via apoptosis [8]. Pleckstrin-homology-like domain family A, member 1 (PHLDA1) is activated in testes in the HSF1-dependent manner and heat-induced cell death has been diminished in the testes of PHLDA1-null mice [9]. Both HSF1 and PHLDA1 are expressed in cryptorchid rat testes in which apoptosis is induced leading to the loss of spermatogenic cells [10]. It has been suggested that the upregulation of PHLDA1 by HSF1 could play a substantial role in promoting heat shock-induced cell death in spermatogenic cells

Methods

Results

Conclusion