Connexin 43 reboots meiosis and reseals blood‐testis barrier following toxicant‐mediated aspermatogenesis and barrier disruption

Abstract

Earlier studies have shown that rats treated with an acute dose of 1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide (adjudin, a male contraceptive under development) causes permanent infertility due to irreversible blood-testis barrier (BTB) disruption even though the population of undifferentiated spermatogonia remains similar to normal rat testes, because spermatogonia fail to differentiate into spermatocytes to enter meiosis. Since other studies have illustrated the significance of connexin 43 (Cx43)-based gap junction in maintaining the homeostasis of BTB in the rat testis and the phenotypes of Sertoli cell-conditional Cx43 knockout mice share many of the similarities of the adjudin-treated rats, we sought to examine if overexpression of Cx43 in these adjudin-treated rats would reseal the disrupted BTB and reinitiate spermatogenesis. A full-length Cx43 cloned into mammalian expression vector pCI-neo was used to transfect testes of adjudin-treated ratsversusempty vector. It was found that overexpression of Cx43 indeed resealed the Sertoli cell tight junction-permeability barrier based on a functionalin vivoassay in tubules displaying signs of meiosis as noted by the presence of round spermatids. Thus, these findings suggest that overexpression of Cx43 reinitiated spermatogenesis at least through the steps of meiosis to generate round spermatids in testes of rats treated with an acute dose of adjudin that led to aspermatogenesis. It was also noted that the round spermatids underwent eventual degeneration with the formation of multinucleated cells following Cx43 overexpression due to the failure of spermiogenesis because no elongating/elongated spermatids were detected in any of the tubules examined. The mechanism by which overexpression of Cx43 reboots meiosis and rescues BTB function was also examined. In summary, overexpression of Cx43 in the testis with aspermatogenesis reboots meiosis and reseals toxicant-induced BTB disruption, even though it fails to support round spermatids to enter spermiogenesis.-Li, N., Mruk, D. D., Mok, K.-W., Li, M. W. M., Wong, C. K. C., Lee, W. M., Han, D., Silvestrini, B., Cheng, C. Y. Connexin 43 reboots meiosis and reseals blood-testis barrier following toxicant-mediated aspermatogenesis and barrier disruption.

Rescue of perfluorooctanesulfonate (PFOS)-mediated Sertoli cell injury by overexpression of gap junction protein connexin 43

“…It was shown that PFOS exerted its effects in Sertoli cells by perturbing F-actin organization in Sertoli cells, thereby disrupting localization of TJ- and basal ES-proteins at the Sertoli cell-cell interface, and associated with a disruption of the GJ-communication function based on a functional dye-transfer FRAP (fluorescence recovery after photobleaching) assay 25 . These phenotypes are somewhat similar to adjudin-induced aspermatogenesis in the testis in vivo 19 20 . We thus sought to examine if overexpression of Cx43 could rescue the PFOS-mediated Sertoli cell injury, because if it could, these findings offer the basis for therapeutic management of PFOS-induced testis injury through gene therapy by overexpressing Cx43, such as the use of nanoparticles that contain mammalian expression vector with the full-length Cx43 cDNA 26 27 28 .…”

“…Overexpression of Cx43 was shown to be able to re-initiate spermatogenesis by re-booting meiosis I/II in adjudin treated rats. For instance, round spermatids were detected in considerable number of tubules vs. adjudin treated rats without Cx43 overexpression in the testis 20 . Detailed analysis of tubules that displayed signs of meiosis in these adjudin treated rats has shown that besides corrective spatiotemporal expression of Cx43 in the seminiferous epithelium similar to normal rat testes, F-actin organization was re-built through proper spatiotemporal expression of actin nucleation protein formin 1 and actin barbed end capping and bundling protein Eps8 20 .…”

“…For instance, round spermatids were detected in considerable number of tubules vs. adjudin treated rats without Cx43 overexpression in the testis 20 . Detailed analysis of tubules that displayed signs of meiosis in these adjudin treated rats has shown that besides corrective spatiotemporal expression of Cx43 in the seminiferous epithelium similar to normal rat testes, F-actin organization was re-built through proper spatiotemporal expression of actin nucleation protein formin 1 and actin barbed end capping and bundling protein Eps8 20 . These changes thus supported proper localization of TJ- (e.g., occludin, ZO-1) and basal ES (ectoplasmic specialization, a testis-specific anchoring junction (for reviews, see refs 21 , 22 , 23 ))- (e.g., N-cadherin, γ-catenin) proteins.…”

Dynein 1 supports spermatid transport and spermiation during spermatogenesis in the rat testis

“…IF was performed using frozen cross sections of testes at 7-m in a cryostat at Ϫ22°C to visualize BTB-associated proteins, such as N-cadherin, ß-catenin, occludin and ZO-1, or actin regulatory proteins Arp3 and Eps8 (48,50). To visualize MTs (e.g., ␣-tubulin), EB1, and Dync1h1, IF was performed using 5-m thick sections of testes fixed in either modified Davidson fixative (for ␣-tubulin or EB1 staining) or Bouin's fixative (for Dync1h1 staining) and paraffin embedded, or fresh Sertoli cells cultured on coverslips (IF) as earlier described (48,50,91). Frozen sections or cells were fixed in 4% PFA or ice-cold methanol for 5-10 min (IF), permeabilized in 0.1% Triton X-100 for 5-10 min (IF).…”

Overexpression of plastin 3 in Sertoli cells disrupts actin microfilament bundle homeostasis and perturbs the tight junction barrier

“…The ORF (open reading frame) of the full-length plastin 3 cDNA clone was amplified from rat Sertoli cell cDNAs reversed transcribed from total RNAs using AccuPrime Pfx DNA polymerase (Invitrogen, Carlsbad, CA), and was cloned into the pCI-neo mammalian expression vector (Promega, Madison, WI) by PCR using the primer pair listed in Table 2, including the corresponding Mul I and Sal I restriction enzyme recognition sites as described. 30,34 This cDNA clone of 1893 bp with start and stop codons was obtained using Sertoli cells total cDNAs, and its identity was confirmed by direct nucleotide sequence analysis at Genewiz, which was identical to GenBank Accession Number: NM_031084.1 for plastin 3 except that nucleotides at 622-624 GGG encoding Gly was GGA (also encoding Gly) in this rat testis clone. The authenticity of this cDNA clone was confirmed in 4 separate cloning and sequencing experiments.…”