Loss of Cx43 in Murine Sertoli Cells Leads to Altered Prepubertal Sertoli Cell Maturation and Impairment of the Mitosis-Meiosis Switch

Abstract

Male factor infertility is a problem in today’s society but many underlying causes are still unknown. The generation of a conditional Sertoli cell (SC)-specific connexin 43 (Cx43) knockout mouse line (SCCx43KO) has provided a translational model. Expression of the gap junction protein Cx43 between adjacent SCs as well as between SCs and germ cells (GCs) is known to be essential for the initiation and maintenance of spermatogenesis in different species and men. Adult SCCx43KO males show altered spermatogenesis and are infertile. Thus, the present study aims to identify molecular mechanisms leading to testicular alterations in prepubertal SCCx43KO mice. Transcriptome analysis of 8-, 10- and 12-day-old mice was performed by next-generation sequencing (NGS). Additionally, candidate genes were examined by qRT-PCR and immunohistochemistry. NGS revealed many significantly differentially expressed genes in the SCCx43KO mice. For example, GC-specific genes were mostly downregulated and found to be involved in meiosis and spermatogonial differentiation (e.g., Dmrtb1, Sohlh1). In contrast, SC-specific genes implicated in SC maturation and proliferation were mostly upregulated (e.g., Amh, Fshr). In conclusion, Cx43 in SCs appears to be required for normal progression of the first wave of spermatogenesis, especially for the mitosis-meiosis switch, and also for the regulation of prepubertal SC maturation.

Connexin43 in Germ Cells Seems to Be Dispensable for Murine Spermatogenesis

“…Thus, aims of the present study were, (1) the establishment of two new conditional KO mouse lines with a GC specific deletion of Cx43 either in premeiotic spermatogonia and early spermatocytes (pGCCx43KO) or in meiotic spermatids (mGCCx43KO) using the Cre/LoxP recombination system to cover the entire postnatal male GC population, (2) investigation of functions of Cx43 in these specific GC populations, and (3) elucidation of possible consequences of the KO of Cx43 in male GC on testicular development and spermatogenesis. Based upon the present data, KO of Cx43 in spermatogonia (pGCCx43KO) or spermatids (mGCCx43KO) did not seem to have dramatic impacts on testicular histology and successful completion of spermatogenesis in adult mice, as it is known for Cx43 in somatic SC [ 31 , 32 , 33 , 35 , 36 , 37 , 44 , 45 , 46 ]. This might be because GC-Cx43 is not essential for successful spermatogenesis or due to compensation by another, yet not identified, Cx in GC forming heterotypic GJ channels with Cx43 in SC.…”

“…Several studies have shown that Cx43 is essential for spermatogenesis [ 27 , 28 , 29 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 44 , 45 , 46 , 47 ]. However, the contribution of known hemichannels provided by GC to this important role could not be determined and consequently is unknown so far.…”

“…The present results suggest that either SC-GC crosstalk via homotypic Cx43-based GJ is not necessary for normal termination of spermatogenesis, or that GC-Cx43 is replaced by another, yet not identified, Cx, resulting in heterotypic GJ channels. It might also be speculated that, while homotypic Cx43 based GJIC between SC is essential for spermatogenesis [ 31 , 32 , 33 , 34 , 35 , 36 , 37 , 44 , 45 , 46 ], SC-GC communication might normally occur via heterotypic GJ composed of Cx43-hemichannels supplied by SC, and connexons of GC assembled by a so far unidentified Cx type. If this was the case, SC-GC communication would not be affected by a GC specific Cx43 KO, but an SC specific deletion of Cx43 would impede the communication between those cell types.…”

“…Previous studies of our research group using SCCx43KO mice showed that deletion of the Gja1 gene in SC affects gene expression in prepubertal mice and, surprisingly, the majority of the significantly regulated genes were GC specific and involved in the regulation of the onset of meiosis [ 37 , 44 ]. This indicates a close connection between SC and GC and that Cx43 in SC participates in the regulation of GC gene expression, GC proliferation and differentiation [ 37 , 44 ]. However, it might also be possible that these changes were caused by missing channel-independent functions of Cx43.…”

Novel Gene Regulation in Normal and Abnormal Spermatogenesis

“…The tight junctions between Sertoli cells in the blood–testis barrier (BTB) are essential for the migration and maturation of male germ cells during spermatogenesis. Conditional knockout of Cx43 (connexin-43) mice assume the downregulated genes critical for mitosis and meiosis, e.g., Stra8 , Dazl , and members of the DM (dsxand map-3) gene family, and the upregulated genes related to Sertoli cell maturation and proliferation [ 81 , 82 ]. The expression levels of cross-epithelial resistance and tight junctions are significantly increased in primary Sertoli cells of mice lacking Cx43 .…”