Expression of Hsf1, Hsf2, and Phlda1 in cells undergoing cryptorchid‐induced apoptosis in rat testes

Abstract

Surgery-induced cryptorchidism, in which the testes are prevented from descending into the scrotal sac, results in testicular germ cell death, and it is commonly used as an experimental tool in the study of spermatogenesis. However, the molecular events underlying the activation of germ cell death remain poorly understood. In the present study, we investigate selective cell loss from cryptorchid rat testis by using DNA flow cytometry and by determining protein and mRNA expression of Hsf1, Hsf2, and Phlda1. The hypo-haploid cell fraction is significantly decreased as early as 3 days after surgical induction of cryptorchidism (from 42.01 ± 5.74% to 15.98 ± 3.88%), followed by a significant decrease in the haploid cell fraction at Day 7. At the latter time point, an apoptotic peak of spermatocytes appears in DNA histograms just before the tetraploid peak; the percentage of aneuploid cells between diploid and tetraploid rises as high as 14.05 ± 2.98% of the total cells in 7-day cryptorchid testis, suggesting that a large number of spermatocytes are undergoing apoptosis. The expression of Phlda1 mRNA is significantly elevated 3 days after induction of cryptorchidism. After 7 days of cryptorchidism, Hsf1 and Phlda1 are strongly expressed in the nucleus and cytoplasm, respectively, of primary spermatocytes. Numerous apoptotic spermatocytes are also observed at this time point. These results suggest that the Hsf1/Phlda1 pathway plays an important role in the apoptosis of primary spermatocytes in cryptorchid testis. We present evidence suggesting that Hsf2 is also involved in germ cell removal in cryptorchid testis.

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

“…Moreover, PHLDA1 did not accumulate after heat shock in any type of spermatogenic cells through the apoptosis was induced and DNA breaks were detected 4-6 h after the treatment, especially in spermatocytes (Figure 2b). On the other hand, in agreement with a previously published report [10], PHLDA1 accumulated in cryptorchid testes 14 days after surgery, also in spermatogenic cells. However, the accumulation of apoptotic cells in cryptorchid testes could be detected earlier than PHLDA1 up-regulation (7 days, but not 14 days after surgery; Figure 2c; see also [7]).…”

“…However, analysis of PHLDA1-induced cell death was not apoptosis-specific in the above study because cells adhering and detaching from dishes were simply counted leaving room for other types of cell death (e.g., anoikis). HSF1/PHLDA1 pathway was also suggested to have a role in cell death of primary spermatocytes in cryptorchid rat testes [10]. However, in this model, the highest level of apoptosis was observed before the accumulation of the PHLDA1 protein.…”

“…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]. Moreover, both HSF1 and PHLDA1 are expressed in cryptorchid rat testes in which apoptosis is induced leading to the loss of spermatogenic cells [10]. Therefore, 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.…”

Altered miRNA profile in testis of post-cryptorchidopexy patients with non-obstructive azoospermia

“…Yin et al revealed that cryptorchidism induced germ cell apoptosis in an experimental mouse model via p53-dependent and p53-independent pathways [ 23 ]. Liu et al also found that the Hsf1/Phlda1 pathway participated in primary spermatocyte apoptosis in surgery-induced cryptorchid testes of rats [ 27 ]. The expression of many apoptosis-related miRNAs was also shown to be altered in post-cryptorchidopexy testicular tissues.…”

Pleckstrin homology-like domain, family A, member 1 (PHLDA1) and cancer

“…In addition, HSF1 is able to directly bind to and activate PHLDA1 in response to heat shock in male germ cells (27). Furthermore, during a time-course induction of cryptorchidism in rat testes, HSF1 and PHLDA1 were strongly expressed, suggesting that the HSF1/PHLDA1 pathways have an important role in the apoptosis of primary spermatocytes in cryptorchid testes (28).…”