Lonidamine-ethyl ester-mediated remodelling of the Sertoli cell cytoskeleton induces phosphorylation of plakoglobin and promotes its interaction with α-catenin at the blood-testis barrier.

Several compounds affect male fertility by disrupting the adhesion of germ cells to Sertoli cells, which results in the release of undeveloped germ cells into the seminiferous tubule lumen that are incapable of fertilising the ovum. Indazole carboxylic acids are one class of compounds exhibiting such effects and they have been investigated as non-hormonal contraceptives for potential human use. The aims of this study were to investigate the effects of lonidamine-ethyl ester, an indazole carboxylic acid, on spermatogenesis and cell junctions, in particular, desmosomes. We found two doses of lonidamine-ethyl ester at 50mg kg-1 to disrupt Sertoli-germ cell adhesion. By light and fluorescent microscopy, pronounced changes were observed in the distribution of actin microfilaments and intermediate filaments, as well as in the localisation of plakoglobin, a protein with structural and signalling roles at the desmosome and adherens junction at the blood-testis barrier. Furthermore, immunoblotting and immunoprecipitation experiments using testis lysates revealed a significant upregulation (P<0.01) of plakoglobin and Tyr-phosphorylated plakoglobin. Co-immunoprecipitation experiments showed an increase in the interaction between plakoglobin and fyn proto-oncogene, an Src family non-receptor tyrosine kinase, after treatment, as well as an increase in the interaction between plakoglobin and α-catenin. Taken collectively, these data indicate that a disruption of Sertoli cell and spermatocyte-spermatid adhesion in the seminiferous epithelium by lonidamine-ethyl ester results in the phosphorylation of plakoglobin, thereby promoting its interaction with α-catenin at the blood-testis barrier.

Effective Delivery of Male Contraceptives Behind the Blood-Testis Barrier (BTB) - Lesson from Adjudin.

The blood-testis barrier (BTB) is one of the tightest blood-tissue barriers in the mammalian body. It divides the seminiferous epithelium of the seminiferous tubule, the functional unit of the testis, where spermatogenesis takes place, into the basal and the adluminal (apical) compartments. Functionally, the BTB provides a unique microenvironment for meiosis I/II and post-meiotic spermatid development which take place exclusively in the apical compartment, away from the host immune system, and it contributes to the immune privilege status of testis. However, the BTB also poses major obstacles in developing male contraceptives (e.g., adjudin) that exert their effects on germ cells in the apical compartment, such as by disrupting spermatid adhesion to the Sertoli cell, causing germ cell exfoliation from the testis. Besides the tight junction (TJ) between adjacent Sertoli cells at the BTB that restricts the entry of contraceptives from the microvessels in the interstitium to the adluminal compartment, drug transporters, such as P-glycoprotein and multidrug resistance-associated protein 1 (MRP1), are also present that actively pump drugs out of the testis, limiting drug bioavailability. Recent advances in drug formulations, such as drug particle micronization (<50 µm) and co-grinding of drug particles with ß-cyclodextrin have improved bioavailability of contraceptives via considerable increase in solubility. Herein, we discuss development in drug formulations using adjudin as an example. We also put emphasis on the possible use of nanotechnology to deliver adjudin to the apical compartment with multidrug magnetic mesoporous silica nanoparticles. These advances in technology will significantly enhance our ability to develop effective non-hormonal male contraceptives for men.

Role of P-glycoprotein at the blood-testis barrier on adjudin distribution in the testis: a revisit of recent data.

The blood-testis barrier (BTB) is one of the tightest blood-tissue barriers in mammals including rodents and humans. It is used to sequester meiosis I and II, postmeiotic spermatid development via spermiogenesis and the release of sperm at spermiation from the systemic circulation, such that these events take place in an immune-privileged site in the adluminal (apical) compartment behind the BTB, segregated from the host immune system. Additionally, drug transporters, namely efflux (e.g., P-glycoprotein) and influx (e.g., Oatp3) pumps, many of which are integral membrane proteins in Sertoli cells at the BTB also work cooperatively to restrict the entry of drugs, toxicants, chemicals, steroids and other xenobiotics into the adluminal compartment. As such, the BTB that serves as an important physiological and selective barrier to protect germ cell development also poses a "hurdle" in male contraceptive development. For instance, adjudin, 1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide, a potential nonhormonal male contraceptive that exerts its effects on germ cell adhesion, most notably at the Sertoli cell-spermatid interface, to induce "premature" germ cell loss from the seminiferous epithelium mimicking spermiation, has a relatively poor bioavailability largely because of the BTB. Since male contraceptives (e.g., adjudin) will be used by healthy men for an extended period of his life span after puberty, a better understanding on the BTB is necessary in order to effectively deliver drugs across this blood-tissue barrier in particular if these compounds exert their effects on developing germ cells in the adluminal compartment. This can also reduce long-term toxicity and health risk if the effective dosing can be lowered in order to widen the margin between its safety and efficacy. Herein, we summarize latest findings in this area of research, we also provide a critical evaluation on research areas that deserve attention in future studies.

Environmental toxicants and male reproductive function.

Environmental toxicants, such as cadmium and bisphenol A (BPA) are endocrine disruptors. In utero, perinatal or neonatal exposure of BPA to rats affect the male reproductive function, such as the blood-testis barrier (BTB) integrity. This effect of BPA on BTB integrity in immature rats is likely mediated via a loss of gap junction function at the BTB, failing to coordinate tight junction and anchoring junction function at the site to maintain the immunological barrier integrity. This in turn activates the extracellular signal-regulated kinases 1/2 (Erk1/2) downstream and an increase in protein endocytosis, destabilizing the BTB. The cadmium-induced disruption of testicular dysfunction is mediated initially via its effects on the occludin/ZO-1/focal adhesion kinase (FAK) complex at the BTB, causing redistribution of proteins at the Sertoli-Sertoli cell interface, leading to the BTB disruption. The damaging effects of these toxicants to testicular function are mediated by mitogen-activated protein kinases (MAPK) downstream, which in turn perturbs the actin bundling and accelerates the actin-branching activity, causing disruption of the Sertoli cell tight junction (TJ)-barrier function at the BTB and perturbing spermatid adhesion at the apical ectoplasmic specialization (apical ES, a testis-specific anchoring junction type) that leads to premature release of germ cells from the testis. However, the use of specific inhibitors against MAPK was shown to block or delay the cadmium-induced testicular injury, such as BTB disruption and germ cell loss. These findings suggest that there may be a common downstream p38 and/or Erk1/2 MAPK-based signaling pathway involving polarity proteins and actin regulators that is shared between different toxicants that induce male reproductive dysfunction. As such, the use of inhibitors and/or antagonists against specific MAPKs can possibly be used to "manage" the illnesses caused by these toxicants and/or "protect" industrial workers being exposed to high levels of these toxicants in their work environment.

Regulation of blood-testis barrier dynamics by desmosome, gap junction, hemidesmosome and polarity proteins: An unexpected turn of events.

The blood-testis barrier (BTB) is a unique ultrastructure in the mammalian testis. Unlike other blood-tissue barriers, such as the blood-brain barrier and the blood-ocular (or blood-retina) barrier which formed by tight junctions (TJ) between endothelial cells of the microvessels, the BTB is constituted by coexisting TJ, basal ectoplasmic specialization (basal ES), desmosomes and gap junctions between adjacent Sertoli cells near the basement membrane of the seminiferous tubule. The BTB also divides the seminiferous epithelium into the apical (or adluminal) and basal compartments so that meiosis I and II and post-meiotic germ cell development can all take place in a specialized microenvironment in the apical compartment behind the BTB. While the unusual anatomical features of the BTB have been known for decades, the physiological function of the coexisting junctions, in particular the desmosome and gap junction, that constitute the BTB was unknown until recently. Based on recently published findings, we critically evaluate the role of the desmosome and gap junction that serve as a signaling platform to coordinate the "opening" and "closing" of the TJ-permeability barrier conferred by TJ and basal ES during the seminiferous epithelial cycle of spermatogenesis. This is made possible by polarity proteins working in concert with nonreceptor protein tyrosine kinases, such as focal adhesion kinase (FAK) and c-Src, at the site to regulate endosome-mediated protein trafficking events (e.g., endocytosis, transcytosis, recycling or protein degradation). These events not only serve to destabilize the existing "old" BTB above preleptotene spermatocytes in transit in "clones" at the BTB, but also contribute to the assembly of "new" BTB below the transiting spermatocytes. Furthermore, hemidesmosomes at the Sertoli cell-basement membrane interface also contribute to the BTB restructuring events at stage VIII of the epithelial cycle. Additionally, the findings that a gap junction at the BTB provides a possible route for the passage of toxicants [e.g., bisphenol A (BPA)] and potential male contraceptives (e.g., adjudin) across the BTB also illustrate that these coexisting junctions, while helpful to maintain the immunological barrier integrity during the transit of spermatocytes, can be the "gateway" to making the BTB so vulnerable to toxicants and/or chemicals, causing male reproductive dysfunction.

Actin-binding protein drebrin E is involved in junction dynamics during spermatogenesis.

The actin-based cytoskeleton plays a critical role in the seminiferous epithelium during spermatogenesis by conferring cell shape, adhesion, structural support and cell polarity to both Sertoli and developing germ cells, which are essential for spermatogonial stem cell renewal, maintenance of the stem cell niche, cell cycle progression, mitosis, meiosis, spermiogenesis and spermiation. However, few functional studies are found in the literature, which explore the functional significance of actin dynamics in these events. This by and large is due to a lack of information on the proteins that regulate actin dynamics. Herein, we report drebrin E is an integrated component of the apical ectoplasmic specialization (apical ES) and the basal ES at the blood-testis barrier (BTB) in the seminiferous epithelium of the adult rat testis. Using immunohistochemistry and dual-labeled immunofluorescence analysis, drebrin E was found to display a stage-specific localization at the apical ES, as well as at the basal ES at the BTB during the seminiferous epithelial cycle of spermatogenesis. Drebrin E was first detected in stage V tubules at the basal ES with the highest expression at the BTB at stages V and VI, but it diminished considerably by stages VII and VIII and was almost non-detectable until stage IV. At the apical ES, drebrin E was also first detected at stage V, surrounding the entire head of the elongating spermatid, but by stage VI its localization had "shifted" to localize most intensely and almost exclusively to the concave side of the spermatid head. In stage VII tubules, drebrin E co-localized with actin, as well as with two other actin regulatory proteins Eps8 (epidermal growth factor receptor pathway substrate 8, an actin capping and bundling protein) and Arp3 (actin-related protein 3, a component of the Arp2/3 complex known to regulate actin nucleation and branching). The localization of drebrin E at the apical ES was compromised following treatment of rats with adjudin, which is known to exert its destructive effects primarily at the apical ES by inducing premature loss of elongating/elongated spermatids from the epithelium, mimicking "spermiation." Instead of being restricted to the concave side of spermatid heads, drebrin E was found to be mis-localized in the seminiferous epithelium of adjudin-treated rats; it was also present on the convex side of elongating spermatids, but these cells were mis-oriented so that their heads no longer pointed toward the basement membrane. The expression of drebrin E by Sertoli cells was also found to be modulated by TGFß3 and TNFα. Since Arp3, but not Eps8, was found to bind drebrin E; and cytokines were also shown to affect the cellular distribution of drebrin E and enhance the interaction between drebrin E and Arp3, these findings illustrate that cytokines may regulate BTB dynamics during the epithelial cycle by recruiting drebrin E and Arp3 to the BTB microenvironment to induce changes in the configuration of actin filament bundles at the basal ES. In summary, these findings illustrate drebrin E is working in concert with Arp3 to regulate actin filament bundles at both the apical and the basal ES in the testis, conferring adhesion and cell polarity at both sites during spermatogenesis.

Adjudin targeting rabbit germ cell adhesion as a male contraceptive: a pharmacokinetics study.

Adjudin (1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide; formerly called AF-2364) has been shown to inhibit spermatogenesis by disrupting anchoring junctions at the Sertoligerm cell interface. This, in turn, leads to germ cell loss from the seminiferous epithelium, and transient infertility. Adjudin's efficacyin inhibiting spermatogenesis, the recovery of spermatogenesis after cessation of the drug, and side effects were examined in adult male Japanese rabbits. The pharmacokinetics profiles of adjudin in rabbits after oral administration and after intravenous injection were compared. Rabbits received 25 mg/kg adjudin once weekly for 4 consecutive weeks either by intravenous injection or by gavage. Vehicle-treated rabbits were used as controls. At 1, 2, 3, 4, and 8 weeks after treatment, testes were removed for microscopic examination to assess the status of spermatogenesis. Four weeks after intravenous cessation of adjudin, the recovery of spermatogenesis also was monitored. Blood was withdrawn after first administration to measure plasma concentrations of adjudin by high-performance liquid chromatography. Four weeks after intravenous treatment, examination of testis sections showed rapid exfoliation of elongated/elongating spermatids and the presence of large multinucleated cells; more than 95% of germ cells were absent from the seminiferous epithelium. Intravenous treatment showed a more severe disturbance of spermatogenesis compared with gavage treatment, which was correlated with bioavailability of the drug. The areas under the curve for intravenous injection and gavage were 20.11 +/- 1.90 and 2.23 +/- 0.45 mg x h x L(-1), respectively. These results illustrate the potential of adjudin as a male contraceptive, and the efficacy is associated with the bioavailability of the drug.

AF-2364 [1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide] is a potential male contraceptive: a review of recent data.

Earlier studies have shown that 1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide (AF-2364) is a potential male contraceptive when administered orally to adult Sprague-Dawley rats. This compound induces reversible germ cell loss from the seminiferous epithelium by disrupting cell adhesion function between Sertoli and germ cells, in particular, elongating/elongate/round spermatids and spermatocytes but not spermatogonia. Thus, this event is accompanied by a transient loss of fertility in treated rats. Once the drug is metabolically cleared, the remaining spermatogonia can begin repopulating the epithelium, and fertility bounces back. In this review, we summarize recent findings regarding the possible use of this drug for male contraception and its mechanism of action in the rat testis. We also provide an update on the efficacy results of using different treatment regimens in adult rats where AF-2364 was administered by gavage vs. intraperitoneal and intramuscular administration. These results have clearly indicated that AF-2364 is indeed a reversible male contraceptive. Furthermore, the tissue distribution in multiple organs and biological fluids using [3H]-AF-2364 is also reviewed. These data have clearly illustrated the low bioavailability of AF-2364 in rats and that this compound is not specifically taken up by any organs including the testis or the epididymis. These summaries are helpful to investigators in the field who seek to understand the molecular mechanism of action of AF-2364 in the rat testis and to explore its possible use for male contraception.