Blockage of testicular connexins induced apoptosis in rat seminiferous epithelium

Abstract

Spermatogenesis, a tightly regulated developmental process of male germ cells in testis, is associated with temporal and spatial expression of gap junction proteins, such as the connexin family members. Perturbation of their expressions may lead to spermatogenic arrest as manifested by disruption of cell-cell interaction. To explore the role(s) of connexins during spermatogenesis, we utilized the small peptide antagonistic approach to specifically deplete connexin 31, connexin 33, and pan-connexin. Three connexin peptides corresponding to the extracellular binding domain of connexin 31 and connexin 33 and to the extracellular conserved domain of connexins were designed and synthesized commercially. Peptides (at single dosage of 0.5, 1, or 2 mg per animal) were injected into rat testes and testes were collected on day 0, 1, 3, 5, 10, 15, and 30 after microinjection. In situ TUNEL assay demonstrated the induction of apoptosis in the testes after pan-connexin peptide treatment in a dose-dependent manner from day 3 and onward. Unlike the pan-connexin peptide, connexin 31 and connexin 33 peptides appeared to have little effect on inducing apoptosis and germ cell loss. CD45 staining also detected the occasional presence of infiltrating lymphocytes in the seminiferous tubules. Accompanied with the apoptotic events, two apoptotic markers, NF-kappaB and caspase 3, demonstrated a general up-regulation in their expressions. In adjacent testis sections, eliminations of connexin 31, 32, and 43 were observed. However, an induction of connexin 33 expression was detected. This suggests the versatility and functional diversity of connexins in the testis. The expression of ZO-1, the only known adaptor of connexins in the testis, was reduced and remained in a low level in the seminiferous epithelium. As such, the alterations of connexins in seminiferous epithelium may induce apoptotic signaling in the testis via the caspase 3 and the NF-kappaB pathway. This demonstrates the significant role of testicular connexins to maintain the survival of germ cells by regulating inter-cellular communications among germ cells and adjacent supporting cells during spermatogenesis. In addition, the inter-relationship between connexins and other junction proteins and associated signaling protein were investigated. After pan-connexin peptide treatment, a dys-localization of N-cadherin, an adherens junction protein, and diminution of occludin, a tight junction protein, level were detected. In addition, inductions of junction regulatory protein, cathepsin L, was observed during the course of peptide-mediated germ cell loss in the testes. In summary, pan-connexin peptide treatment triggered apoptosis and germ cell loss in the testes. This event influenced the localization and expression of different junction proteins and junction-associated protein in the testes.

Blood–testis barrier and Sertoli cell function: lessons from SCCx43KO mice

“…This hypothesis is in agreement with the previous morphological studies in continual (guinea pig) and seasonal breeder (mink) testes, which suggest that connexin43 (CX43) between SC participates in the formation and regulation of the BTB (Pelletier 1995). Recently, it has been reported that the blockage of CX, by using a pan-CX peptide that recognizes all characterized CX within the testis, concomitantly leads to diminished OCCLUDIN and upregulated N-CADHERIN expression (Lee et al 2006).…”

A mitochondrial mechanism is involved in apoptosis of Robertsonian mouse male germ cells

“…Some authors have found that immature spermatogonia show features of apoptosis, while more mature spermatocytes do not (Allan et al 1987, Russell 1999; however, others have found that in the first wave of spermatogenesis primarily mid-pachytene spermatocytes die by apoptosis (Jahnukainen et al 2004). Cell death is, therefore, a normal process during spermatogenesis, limiting the number of germ cells to ensure that Sertoli cells can provide enough nutrients to nurture the continuous generation of germ cells (Lee et al 2006). The Fas system has been shown to be involved in the regulation of germ cell apoptosis in the rat testis.…”

Nitric Oxide and Cyclic Nucleotides: Their Roles in Junction Dynamics and Spermatogenesis

“…When rats were treated with a blocking connexin peptide, disruption of connexin functions using pan-connexin peptide renders the disruption of occludin-based TJ, but not N-cadherin-based AJ in the testes. 23 This proves the vulnerability of the occludin-associated complex following a dysfunction of connexin-based GJ, since pan-connexin peptide with sequence conserved among all connexins virtually blocks all connexin-associated functions in the testes. 23 In addition, the structural association of γ-catenin, an AJ adaptor, and ZO-1, a TJ adaptor, is weakened after treatment of rats using adjudin, which is shown to have a role in disturbing the AJ without compromising the TJ in the testes.…”

“…23 This proves the vulnerability of the occludin-associated complex following a dysfunction of connexin-based GJ, since pan-connexin peptide with sequence conserved among all connexins virtually blocks all connexin-associated functions in the testes. 23 In addition, the structural association of γ-catenin, an AJ adaptor, and ZO-1, a TJ adaptor, is weakened after treatment of rats using adjudin, which is shown to have a role in disturbing the AJ without compromising the TJ in the testes. 3,24 These findings substantiate the significance of adaptors that mediate cross-talk between different junction types in the testes.…”

“…These pathways are probably inter-connected and mediated by junction proteins, such as connexins, during apoptosis in the testes. 23 There are reports in the literature illustrating an excessive NO level can directly trigger massive germ cell apoptosis in the testes. These are clearly depicted in the artificial spermatic vessel ligation model 55 and in the mouse model of congenital cryptorchidism, 56 both of which were shown to elevate NO testicular levels and uncontrolled germ cell apoptosis.…”