The effects of green tea, green rooibos and their major flavonoids (EGCG and aspalathin) on testicular cell health in vitro

Abstract

The testes play a central role in the male reproductive system, as they represent the sites of male sex steroidogenesis and of spermatogenesis. Leydig cells, located at the testicular interstitium, produce predominantly testosterone upon stimulation with either chorionic gonadotropin (CG) or luteinising hormone by a series of enzymatic modifications of cholesterol. Sertoli cells respond to testosterone and follicle stimulating hormone to secrete inhibin and facilitate spermatogenesis by additional activities like maintaining Sertoli cell barrier (SCB) integrity and lactate secretion. Ultimately the Leydig cells, Sertoli cells etc. all work together to confer male fertility. However, infertility occurs globally; leading to the pursuit of treatment, including herbal medicines. Tea and rooibos are popular health drinks with global reach. Both have a green/unfermented form, which are said to possess potent health-beneficial properties. Polyphenols, and especially the flavonoids: epigallocatechin gallate (EGCG, from green tea) and aspalathin (from green rooibos), are held as primarily responsible for their health benefits. These flavonoids are known antioxidants that can interact with proteins, carbohydrates and fats, making them bioactive compounds of interest to health sciences. However, research on the effects of these teas and flavonoids on male reproduction are scarce and sometimes conflicting. Hence, this thesis aimed to determine some of the mechanisms by which green tea, green rooibos, EGCG and aspalathin affect model Leydig and Sertoli cells in vitro. The murine TM3 (Leydig) and TM4 (Sertoli) cell lines were cultured in vitro and exposed to varying concentrations of green tea, green rooibos, EGCG or aspalathin for 24 hours. Thereafter, the cells were assayed for oxidoreductase activity (by MTT: 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), morphological alterations (light microscopy), mitochondrial membrane potential (ΔΨm by tetramethylrhodamine ethyl ester) and reactive oxygen species (by chloromethyl 2',7'-dichlorodihydrofluorescein diacetate). TM3 cells, as well as boar testes ex vivo, were assayed for hCG-stimulated and unstimulated testosterone secretion. TM3 cells were assayed for mRNA expression of selected genes involved in steroidogenesis; namely Lhcgr, Star, Tspo, Cyp11a1, Cyp17a1 and Hsd17b3, as well as for Gapdh as a housekeeping gene. TM4 cells were also assayed for inhibin B secretion, lactate secretion and SCB integrity (by transepithelial electrical resistance). Within the tested concentration range of the teas and flavonoids used in this study, no cytotoxic effects were observed in Leydig or Sertoli cells. The experimental data further suggest that both green tea and green rooibos increase mitochondrial activity, resulting in lower cytosolic NADH, consequently decreasing oxidoreductase activity of Sertoli cells. Furthermore, lactate secretion was slightly reduced in Sertoli cells exposed to the teas. Contrary to this, their major flavonoids, EGCG and aspalathin did not exert the same responses. Sertoli cells exposed to EGCG or aspalathin showed a significant decrease in oxidoreductase activity without affecting lactate secretion or the SCB. In Leydig cells, both EGCG and aspalathin induced slight decreases in ΔΨm without affecting oxidoreductase activity. However, the flavonoids altered steroidogenic gene expression. EGCG increased both basal and hCG-stimulated Tspo expression, as well as stimulated Star expression. In contrast, aspalathin did not affect Star expression, but increased both basal and stimulated Tspo expression. All in all, these data suggest that both EGCG and aspalathin exerted pro-steroidogenic effects on Leydig cells. The murine TM3 (Leydig) and TM4 (Sertoli) cell lines are often used to investigate the effects of plant extracts on testicular functions. Several authors reported testosterone secretion in TM3 cells, and the TM3 cell population used here also proved to produce testosterone before the start of this study. However, in the interim, the cell physiology seems to have changed. As it turned out, both the TM3 and TM4 cell lines seemed to display a divergent physiology. The mRNA from some critical steroidogenic genes (i.e. Lhcgr, Cyp11a1 and Hsd17b3) could not be detected in the TM3 cells via PCR. That could explain why those cells were unresponsive to hCG. The TM4 cells did not secrete detectable levels of inhibin B either, indicating aberrant physiology for Sertoli cells.