Two New Male Contraceptives Exert Their Effects by Depleting Germ Cells Prematurely from the Testis1

Abstract

The three currently available male contraceptive approaches are 1) the barrier method such as the condom, 2) hormonal methods by disrupting the pituitary-testicular axis so as to impair spermatogenesis, and 3) immunological methods by preparing vaccines against male-specific antigens. We hereby describe an alternative approach in which attachments of developing germ cells onto the seminiferous epithelium are disrupted, thereby inducing their premature release into the tubular lumen. This in turn leads to infertility. A panel of analogues based on the core structure of 1-(2,4-dichlorobenzyl)-indazole-3-carboxylic acid was synthesized. These compounds were subjected to an in vivo screening assay assessing their effects in inducing the expression of testin, a testicular marker whose expression correlates with the integrity of Sertoli-germ cell junctions. An induction of testin expression in the testis signifies a disruption of Sertoli-germ cell junctions that is followed by depletion of germ cells from the seminiferous epithelium. Two compounds, namely 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide (AF-2364) and 1-(2,4-dichlorobenzyl)-indazole-3-acrylic acid (AF-2785), were identified that caused detachment of germ cells, in particular round and elongated spermatids, from the epithelium inducing their premature release into the tubular lumen as confirmed by histological analysis. Adult rats receiving several oral doses of either one of these compounds became infertile within 3-7 wk after the epididymal sperm reserve was exhausted. Depending on the dosing of the administered compound, rats became infertile for 4-14 wk before their fertility gradually bounced back, illustrating the reversibility and efficacy of these new compounds. Also, these compounds did not appear to impair the hypothalamus-pituitary-testicular axis because the serum levels of LH, FSH, and testosterone of the treated animals did not change significantly when compared to control rats. In addition, results of serum microchemistry illustrate that liver and kidney function was not affected in animals treated with both compounds.

Blood–testis barrier dynamics are regulated by an engagement/disengagement mechanism between tight and adherens junctions via peripheral adaptors

“…One can argue that the altered localization of TJ and AJ proteins during Adjudin-induced germ cell loss as reported here is the result of widening of interstitial space between adjacent tubules that have extensive germ cell loss and͞or accumulation of interstitial fluid in the interstitium. These possibilities are supported in part by the histological findings consistent with several earlier reports (17,25,31). Furthermore, this significant loss of germ cells from the epithelium can increase the relative contribution of proteins by Sertoli cells and͞or other somatic cells in the samples being analyzed by immunoblottings that can artificially account for protein up-regulation.…”

“…Adult rats (n ϭ 4 rats per time point; Ϸ300 g of body weight) were fed with a single dose of Adjudin at 50 mg͞kg of body weight (17,18). Control rats received vehicle only [0.5% (wt͞vol) methylcellulose in water].…”

Cellular localization of sphingomyelin synthase 2 in the seminiferous epithelium of adult rat testes

“…is an analog of lonidamine that was selected from two dozen new compounds in earlier studies from our laboratory (Cheng et al 2001), which is known to induce adherens junction disruption in adult rat testes (for a review, see Mruk & Cheng 2004b). Recent studies have shown that Adjudin perturbed Sertoli-germ cell adhesion in the seminiferous epithelium of adult rat testes by exerting its effects most predominantly at the apical ectoplasmic specialization (apical ES) (note: apical ES is a testis-specific cell-cell actin-based AJ type) at the Sertoli cell-elongating/elongate spermatid interface, to be followed by disruption of junctions at the Sertoli cellspermatocyte interface (Cheng & Mruk 2002, Chen et al 2003.…”

“…Recent studies have shown that Adjudin perturbed Sertoli-germ cell adhesion in the seminiferous epithelium of adult rat testes by exerting its effects most predominantly at the apical ectoplasmic specialization (apical ES) (note: apical ES is a testis-specific cell-cell actin-based AJ type) at the Sertoli cell-elongating/elongate spermatid interface, to be followed by disruption of junctions at the Sertoli cellspermatocyte interface (Cheng & Mruk 2002, Chen et al 2003. Apparently, junctions at the Sertoli cell-spermatogonia were not affected, since if spermatogonia were depleted from the epithelium, the transient infertility induced by Adjudin treatment would have been irreversible (Cheng et al 2001; for a review, see ). As such, the Adjudininduced germ cell loss from the epithelium in adult rats has been used as an in vivo model to study anchoring junction dynamics pertinent to spermatogenesis .…”

“…through gavage would induce extensive AJ restructuring, leading to germ cell depletion from the seminiferous epithelium, most notably elongating/elongate spermatids, to be followed by round spermatids and spermatocytes (Cheng et al 2001, Chen et al 2003. Interestingly, while SMS2 is not an anchoring junction protein, studies by immunoblotting have shown that a surge in SMS2 protein level was detected as early as 4 h after Adjudin treatment which persisted until day 4 when most germ cells had dislodged from the epithelium, and its level declined rapidly thereafter, possibly as a result of germ cells loss since SMS2 is restricted to germ cells in the testis (Fig.…”