Adjudin disrupts spermatogenesis via the action of some unlikely partners

Abstract

Adjudin, 1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide (formerly called AF-2364), is a potent analog of lonidamine [1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid] known to disrupt germ cell adhesion, most notably elongating and elongated spermatids, in the seminiferous epithelium of adult rat testes and thus, leads to infertility in rats. Since the population of spermatogonia and spermatogonial stem cells (SSCs) in the seminiferous tubules is not significantly reduced by the treatment of rats with adjudin, adjudin-induced infertility is highly reversible, which enables reinitiation of spermatogenesis and germ cell re-population of the voided seminiferous epithelium. Furthermore, adjudin appears to exert its effects at the testis-specific atypical adherens junction (AJ) type known as ectoplasmic specialization (ES), most notably the apical ES at the Sertoli cell-spermatid interface. Thus, the hypothalamic-pituitary-gonadal axis is not unaffected and systemic side-effects are minimal. This also makes adjudin a potential candidate for male contraceptive development. Herein, we critically evaluate recent findings in the field and provide an updated model regarding the mechanism underlying adjudin-induced apical ES disruption. In short, adjudin targets actin filament bundles at the apical ES, the hallmark ultrastructure of this testis-specific junction type not found in any other epithelia/endothelia in mammals, by suppressing the expression of Eps8 (epidermal growth factor receptor pathway substrate 8), an actin capping protein that also plays a role in actin bundling, so that actin filament bundles can no longer be maintained at the apical ES. This is concomitant with a mis-localization of Arp3 (actin-related protein 3, a component of the Arp2/3 complex that induces actin nucleation/branching) recruited by drebrin E, causing "unwanted" actin branching, further destabilizing actin filament bundles at the apical ES. Additionally, adjudin blocks the expression of PAR6 (partitioning defective protein 6) and 14-3-3 (also known as PAR5) considerably at the apical ES, disrupting the homeostasis of endocytic vesicle-mediated protein trafficking, which in turn leads to an increase in protein endocytosis. The net result of these changes destabilizes cell adhesion and induces degeneration of the apical ES, causing premature release of spermatids, mimicking spermiation.

Breast cancer resistance protein (Bcrp) and the testis—an unexpected turn of events

“…Our interest in studying drug transporters stems from our investigation of developing adjudin, 1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide, into a male contraceptive. [13][14][15] Although this compound is highly potent at disrupting germ cell adhesion in the testes, the drug was shown to have very limited bioavailability following oral administration. 16 Success in developing this compound into a male contraceptive relies heavily on our ability to prepare a better formulated version that is both economical to manufacture and that requires a low efficacy dose.…”

Germline deletion of huntingtin causes male infertility and arrested spermiogenesis in mice

“…7D). In particular, the markedly down-expressed proteins upon Htt depletion, such as ODF1, Smcp, ACRBP, CLIP1, EPS8, TYRO3 and BAK1, have been reported to be directly linked to spermatogenesis and male fertility (Baba et al, 1994;Oldereid et al, 2001;Nayernia et al, 2002;Akhmanova et al, 2005;Xiong et al, 2008;Cheng et al, 2011;Yang et al, 2012). Thus, knockout of Htt might impair spermatogenesis through altering the expression of these proteins.…”

Toxicants target cell junctions in the testis: Insights from the indazole-carboxylic acid model

“…50,69 Treatment of rats with adjudin was found to down-regulate Eps8 expression at the apical ES in stage VI-VII tubules. 69,71 Additionally, branched actin-inducing protein Arp3, which effectively turns bundled microfilaments to an unbundled/branched configuration is highly expressed at the apical ES but confined to the concave (ventral) side of spermatid Figure 2. Changes in the seminiferous epithelium of testes following treatment of adult rats with adjudin (50 mg/kg b.w., by oral gavage).…”

“…Studies have shown that the changes at the ES regarding the spatiotemporal expression of Eps8 and Arp3 during the epithelial cycle of spermatogenesis are mediated, at least in part, via the action of FAK 72 that serves as the molecular switch that regulates the organization of actin microfilaments at the Sertoli cell-spermatid interface. 50,71 In short, adjudin perturbs the spatiotemporal expression of these actin regulatory proteins, thereby disrupting the proper organization of F-actin by compromising the conversion of actin microfilaments between their "bundled" to "un-bundled/branched" configuration. This destabilizes the apical ES, leading to its disruption which is accompanied by the premature loss of spermatids from the seminiferous epithelium.…”