A potential novel mechanism involving connexin 43 gap junction for control of sertoli cell proliferation by thyroid hormones

Abstract

There is strong evidence that thyroid hormones through triiodothyronine (T3) regulate Sertoli cell proliferation and differentiation in the neonatal testis. However, the mechanism(s) by which they are able to control Sertoli cell proliferation is unclear. In the present study in vivo approaches (PTU-induced neonatal hypothyroidism known to affect Sertoli cell proliferation) associated with in vitro experiments on a Sertoli cell line were developed to investigate this question. We demonstrated that the inhibitory effect of T3 on Sertoli cell growth, analyzed by evaluating DNA-incorporated [3H] thymidine, was associated with a time and dose-dependent increase in the levels of Cx43, a constitutive protein of gap junctions, known to participate in the control of cell proliferation and the most predominant Cx in the testis. These Cx43 changes were associated with increased gap junction communication measured by gap FRAP. Consistent with these results two specific inhibitors of gap junction coupling, AGA and oleamide, were able to significantly reverse the T3 inhibitory effect on Sertoli cell proliferation. The present data also revealed a nongenomic effect of T3 on Cx43 Sertoli cells that was evidenced by a rapid up-regulation of gap junction plaque number as identified in Cx43-GFP transfected cells exposed to the hormone. This process appears mediated through actin cytoskeleton since incubation of the cells with cytochalasin D totally reversed the T3 stimulatory effect on Cx43-GFP gap junction plaques. Based on these data, we propose a working hypothesis in which Cx43 could be an intermediate target for T3 inhibition of neonatal Sertoli cell proliferation.

Connexin 43 is critical to maintain the homeostasis of the blood–testis barrier via its effects on tight junction reassembly

“…Sertoli cells cultured at 0.15 × 10 6 cells/cm 2 for 2 d on a Matrigel-coated glass-bottom dish were used for fluorescence recovery after photobleaching (FRAP) analysis of calcein transfer (further details in SI Materials and Methods). This assay assesses the degree of gap junction communication by the intercellular transfer of calcein (32). Cells were labeled with 5 μM calcein acetoxymethyl ester (AM) for 30 min at 35°C.…”

Testicular connexin 43, a precocious molecular target for the effect of environmental toxicants on male fertility

“…Indeed, it is recognized that thyroid hormones, with FSH and androgens, participate in the control of neonatal Sertoli cell proliferation and differentiation, and that this control is partly exerted through testicular Cx43. 147,148 Persistent reduced thyroxine levels, after PCB 153 (2,2',4,4',5,5'-hexachlorobiphenyl) exposure, disturbs perinatal Cx43 Sertoli cell expression and subsequently impairs spermatogenesis. 106 Altogether, these data argue for Cx43 as one of the major molecular target for toxicants in the perinatal testis.…”

Molecular reorganization of Cx43, Zo-1 and Src complexes during the endocytosis of gap junction plaques in response to a non-genomic carcinogen

“…When indicated, cells were also transfected with an expression plasmid for Rab5a-wtmRFPC1 (gift from M. Zerial, Max Planck Institut, Dresden, Germany). Transfections were performed using Lipofectamin (Invitrogen) according to the manufacturer's instructions, as recently described (Gilleron et al, 2006). To induce internalization of gap junction plaques, Cx43-GFP transfected Sertoli cells were incubated in the presence or absence of 50 μM γ-hexachlorocyclohexane (HCH), an organochlorine compound that has previously been reported to efficiently stimulate Cx43 internalization (Guan and Ruch, 1996;Defamie et al, 2001).…”

“…Functionality of gap junction channels was evaluated by analyzing the passage of a fluorescent dye through these junctions as previously described (Gilleron et al, 2006). Briefly, cells were incubated with 5 μM of calcein-AM (Invitrogen SARL, Cergy Pontoise, France) in DMEM for 30 minutes at 32°C.…”