Secretion of glutathione S-transferase isoforms in the seminiferous tubular fluid, tissue distribution and sex steroid binding by rat GSTM1

Abstract

The seminiferous tubular fluid (STF) provides the microenvironment necessary for spermatogenesis in the adluminal compartment of the seminiferous tubule (ST), primarily through secretions of the Sertoli cell. Earlier studies from this laboratory demonstrated the presence of glutathione S-transferase (GST) in STF collected from adult rat testis and in the spent media of ST cultures. This study describes the cellular source, isoform composition and possible function of GSTs in the STF. The major GST isoforms present in STF in vivo share extensive N-terminal similarity with rat GSTM1 (rGSTM1), rGSTM2, rGSTM3 and rGST-Alpha. Molecular masses of rGSTM2, rGSTM3 and rGST-Alpha from liver and testis sources were similar, unlike STF-GSTM1, which was larger by 325 Da than its liver counterpart. Peptide digest analysis profiles on reverse-phase HPLC between liver and STF isoforms were identical, and N-terminal sequences of selected peptides obtained by digestion of the various isoforms were closely similar. The above results confirmed close structural similarity between liver and STF-GST isoforms. Active synthesis and secretion of GSTs by the STs were evident from recovery of radiolabelled GST from the spent media of ST cultures. Analysis of secreted GST isoforms showed that GST-Alpha was not secreted by the STs in vitro, whereas there was an induction of GST-Pi secretion. Detection of immunostainable GST-Mu in Sertoli cells in vitro and during different stages of the seminiferous epithelium in vivo, coupled with the recovery of radiolabelled GST from Sertoli cell-culture media, provided evidence for Sertoli cells as secretors of GST. In addition, STF of 'Sertoli cell only' animals showed no change in the profile of GST isoform secretion, thereby confirming Sertoli cells as prime GST secretors. Non-recovery of [35S]methionine-labelled GSTs from germ cell culture supernatants, but their presence in germ cell lysates, confirm the ability of the germ cells to synthesize, but not to release, GSTs. Functionally, STF-GSTM1 appeared to serve as a steroid-binding protein by its ability to bind to testosterone and oestradiol, two important hormones in the ST that are essential for spermatogenesis, with binding constants of <9.8x10(-7) M for testosterone and 9x10(-6) M for oestradiol respectively.

Effects of Male Reproductive Toxicants on Gene Expression in Rat Testes

“…It was found in the present cDNA microarray analysis that the gene expression of Sertoli-germ cells adhesion molecules such as cadherin (Johnson et al, 2000), fibronectin (Schaller et al, 1993), and integrin (Siu et al, 2003) was decreased in the 2,5-HD high-dose group. Furthermore, the genes that are expressed in Sertoli cells such as IGFBP-3 (Smith et al, 1990), ferritin (Carreau, 1996), epidermal growth factor receptor (Suarez-Quian et al, 1994) and GST-pi (Mukherjee et al, 1999) were altered. Expression changes of these genes might underlie the toxicity of 2,5-HD observed in Sertoli cells.…”

“…HSP70-2 is a molecular chaperon for meiosis in spermatogenesis and knockout mice lacking this gene are infertile (Eddy, 1999). GST-pi is contained in seminiferous tubular fluid (Aravinda et al, 1995), and this fluid provides the microenvironment necessary for spermatogenesis (Mukherjee, et al, 1999). Therefore, alterations of the expression of these molecules may lead to anomalous spermatogenesis.…”

OXIDATIVE STRESS AND REPRODUCTIVE FUNCTION: The protection of mammalian spermatozoa against oxidative stress

“…Different glutathione-S-transferases (GSTs) isoforms are present in the testes, epididymides, and spermatozoa from different mammalian species, including humans, goats, boars, buffalos, and rodents (Veri et al 1993, Gopalakrishnan et al 1998, Mukherjee et al 1999, Raijmakers et al 2003, Kumar et al 2014, Llavanera et al 2020. GSTs are being discovered as crucial elements in the antioxidant response to sustain male fertility.…”

Ultrastructural localization of 28 kDa glutathione S-transferase in adult Clonorchis sinensis

“…In the present study, 28GST was shown to be localized most densely in sperm tails of of C. sinensis. Isoform of class Mu GST is present in human testes (Hussey and Hayes, 1993) and abundant in fluid of seminiferous tubules of rat testes (Mukherjee et al, 1999). Sertoli cells are prime secretors of Mu GSTs in rat testes.…”