Anchoring junctions as drug targets: role in contraceptive development

Abstract

In multicellular organisms, cell-cell interactions are mediated in part by cell junctions, which underlie tissue architecture. Throughout spermatogenesis, for instance, preleptotene leptotene spermatocytes residing in the basal compartment of the seminiferous epithelium must traverse the blood-testis barrier to enter the adluminal compartment for continued development. At the same time, germ cells must also remain attached to Sertoli cells, and numerous studies have reported extensive restructuring at the Sertoli-Sertoli and Sertoli-germ cell interface during germ cell movement across the seminiferous epithelium. Furthermore, the proteins and signaling cascades that regulate adhesion between testicular cells have been largely delineated. These findings have unveiled a number of potential "druggable" targets that can be used to induce premature release of germ cells from the seminiferous epithelium, resulting in transient infertility. Herein, we discuss a novel approach with the aim of developing a nonhormonal male contraceptive for future human use, one that involves perturbing adhesion between Sertoli and germ cells in the testis.

FIG. 1

Schematic drawing illustrating the molecular architecture of the apical ectoplasmic specialization in the adult rat testis. Ectoplasmic specialization function is constituted by at least four multiprotein complexes: 1) cadherin-catenin, 2) nectin-afadin, 3) integrin-laminin, and 4) vezatin-myosin, and these link indirectly to the cytoskeleton. As discussed in this review, germ cell adhesion to Sertoli cells is regulated by many molecules, including phosphatases and kinases, cytokines, and GTPases. Recent studies have also suggested that there is cross-talk between different multiprotein complexes at the anchoring junction, which is likely to contribute significantly to the regulation of Sertoli-germ cell adhesion in the testis. Thus, many of these proteins can become “druggable” targets for nonhormonal male contraceptive development. This figure was prepared based on several recently published original research and review articles in the field, and these are cited throughout the text. Molecules that are seemingly important in cell adhesion but that have yet to be studied in detail in the testis are denoted with a question mark.

FIG. 2

The desmosome-like junction in the adult rat testis. A, schematic illustration showing constituent proteins of the desmosome junctions. The desmosome junction uses intermediate filaments for cytoplasmic attachment. B, electron micrograph showing typical ultrastructural features of the desmosome-like junction in the testis. This junction type is characterized by the presence of electron dense material (see arrows) between two apposing Sertoli cell (SC) plasma membranes. They were called desmosome-like junctions because they shared properties of both desmosomes and gap junctions (Russell, 1977a). Bar in B, 0.25 µm.

FIG. 3

Blood-testis barrier in the adult rat testis. Electron micrograph of the blood-testis barrier present between adjacent Sertoli cells (SC) near the basement membrane (bm, see arrow and asterisks), which is a modified form of the ECM that appears as an amorphous substance. Underneath this lies a layer of type I collagen, and cross-sectioned collagen bundles are clearly visible. This is followed by peritubular myoid cells (pmc). The blood-testis barrier is characterized largely by the coexistence of tight junctions (tj; see green bracket), basal ectoplasmic specializations (es; see red bracket), and desmosome-like junctions (dj; see blue brackets). The basal ectoplasmic specialization is typified by the presence of actin filament bundles (see red asterisks) sandwiched between the endoplasmic reticulum (er) and the Sertoli cell plasma membrane. Desmosomes are typified by electron dense material present between two adjacent Sertoli cells, whereas tight junctions are characterized by “kisses,” regions of close contact between two apposing Sertoli cell plasma membranes (see green arrowheads). Bar, 0.75 µm. [Reproduced from Sarkar O, Mathur PP, Cheng CY, and Mruk DD (2008) Interleukin-1 alpha (IL1A) is a novel regulator of the blood-testis barrier in the rat. Biol Reprod 78:445–454. Copyright © 2008 Society for the Study of Reproduction. Used with permission.]

FIG. 4

Cross-section of a seminiferous tubule illustrating the intimate relationship between Sertoli and germ cells in the seminiferous epithelium of an adult rat testis. All cells in the seminiferous epithelium sit on top of the tunica propria (tp). A Sertoli cell nucleus (SC) is visualized in this micrograph and located basally within the seminiferous epithelium. It is noteworthy that each Sertoli cell has the ability to support ~30 to 40 germ cells at various stages of development, including spermatogonia (sg), pachytene spermatocytes (p.sp), round (r.sp), and elongating (es) spermatids. During spermiation, elongated spermatids release into the tubule lumen and then travel to the epididymis for further development. The blood-testis barrier (see Fig. 3), which is formed by adjacent Sertoli cells, physically divides the seminiferous epithelium into basal and adluminal compartments. Bar, 12 µm.

FIG. 5

Stages of the seminiferous epithelial cycle in the adult rat testis. Each of the 14 stages shown consists of three parts: 1) a cross-section of the seminiferous epithelium (paraffin-embedded testes) stained with hematoxylin and eosin, 2) an illustration of the different types of germ cells associating with that particular stage, and 3) the estimated length of each stage (in hours). Stages II and III are the most difficult to distinguish correctly. Thus, the duration of these two stages has been combined into a single time point. One complete seminiferous epithelial cycle in the rat lasts for 12.9 days. Spermatogonial development is subdivided into type A1–4, intermediate (Int), and type B (B). Spermiogenesis is subdivided into steps 1 to 19 to more accurately define the morphological changes that occur in spermatids during development. In the figure, stages VIII to XI have been enclosed in red boxes to define the approximate time when leptotene spermatocytes traverse the blood-testis barrier, entering into the adluminal compartment for further development. P, pachytene spermatocyte; PL, preleptotene spermatocyte; L, leptotene spermatocyte; Z, zygotene spermatocyte; D, diplotene spermatocyte; SS, secondary spermatocyte; M1, meiosis I; M2, meiosis II. [Prepared based on earlier reports in Stages 2.2, a graphical program designed by Drs. Rex Hess and David Scott (University of Illinois, Urbana-Champaign, IL), and Russell et al., 1990.]

FIG. 6

Apical ectoplasmic specialization in the adult rat testis. Electron micrograph of the apical ectoplasmic specialization (es, see brackets) between a Sertoli cell (SC) and elongating spermatid consisting of hexagonally arranged bundles of actin filaments (see arrowheads) sandwiched between the Sertoli cell membrane and flattened cisternae of endoplasmic reticulum (see asterisks). Also shown are microtubules (see arrows). Bar, 0.4 µm.

FIG. 7

Chemical structure of adjudin. Adjudin is a derivative of lonidamine [1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid], an anticancer drug (Silvestrini et al., 1984).

FIG. 8

A study to examine the effects of adjudin in the epididymis. A single oral dose of adjudin at 50 mg/kg b.wt. was administered to adult rats. On days 2, 4, and 8, epididymides were removed, dissected into three segments [caput (initial segment), corpus (middle segment), and cauda (final segment)] and processed for histological analysis after hematoxylin & eosin staining using paraffin sections. By days 2 and 4 after treatment, immature germ cells such as spermatocytes and early spermatids (see arrowheads in B) were detected in the caput (A–C), but by day 8, these cells had depleted this segment of the epididymis (D). On days 2 and 4, normal epididymal spermatozoa were found in the corpus (E) and cauda (F), and apparently their function was unaffected by adjudin, because all animals remained fertile until the epididymis became devoid of all spermatozoa by ~30 days after treatment (Cheng et al., 2005a). By day 8, however, immature germ cells were seen in the cauda mixed together with normal epididymal spermatozoa (G). In addition, adjudin did not affect adhesion between epididymal epithelial cells. Bars, A and C to G, 120 µm; B, 60 µm.

FIG. 9

Ultrastructural changes in the seminiferous epithelium of the adult rat testis after treatment of animals with a single oral dose of adjudin at 50 mg/kg b.wt. This is an electron micrograph of the seminiferous epithelium, and Sertoli and germ cells, namely spermatogonia (sg), are seen lying on top of the tunica propria (tp). The blood-testis barrier (see boxed area) appears to be normal 1 week after administration of adjudin. However, many intercellular spaces (see asterisks) are seen between a pachytene spermatocyte (sp) and a Sertoli cell, illustrating loss of adhesion. ld, lipid droplet; n, Sertoli cell nucleus. Bar, 6 µm.

FIG. 10

Estimation of the half-time of disappearance of [³H]adjudin in adult rats after i.v. administration. The half-time of disappearance of adjudin was estimated in adult rats (~300 g b.wt., n = 3 per time point) after i.v. administration of [³H]adjudin. In brief, [³H]adjudin, [indazole-5,7-³H(N)]-1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide, was purchased (PerkinElmer Life and Analytical Sciences, Waltham, MA). The purity of [³H]adjudin was confirmed by 1) high-performance liquid chromatography using a Zorbax C18 reversed-phase column, which had an identical retention time when both ³H-labeled and unlabeled adjudin were injected onto the column simultaneously or separately with the eluents monitored by UV absorbance at 210 nm or spectrophotometry using a β-counter, 2) mass spectrometry, and 3) elemental analysis. The t_1/2 of adjudin in the systemic circulation of adult rats was estimated by injecting ~3 × 10⁶ cpm of [³H]adjudin in a sample volume of 50 µl of phosphate-buffered saline via the jugular vein. An aliquot of blood was collected from the tail vein from each rat in this experiment at 0.5, 1, 3.5, 7, 10, 24, 48, 76, and 120 h after administration of [³H]adjudin and was allowed to clot. Serum was obtained by centrifugation for radioactivity determination. The t_1/2 was determined using nonlinear least-squares curve-fitting techniques to fit [³H]adjudin levels in blood as a function of time to a multiexponential function consisting of one to four terms of the following equation: Y(t) = ΣA_ie^−Bit, where Y(t) is the response variable (in this case, the level of [³H]adjudin obtained in the blood sample). Data were fitted using a computer program based on code implementing the Marquardt algorithm to minimize χ² (Bevington, 1969). Because data exhibited nonuniform variation, they were weighted as 1/σ², where σ was determined from samples of three different animals at each time point. The number of exponentials fitted to experimental data was determined as the number that minimized χ². Estimates were obtained for the parameters σA_i and σB_i, as well as their uncertainties A_i and B_i, which were derived from the diagonal terms in the error matrix generated during the fitting procedure. Note that the estimates for the parameter uncertainties did not take into account covariance terms and, as a result, tended to be underestimated. However, this analysis yielded the best estimate on the disappearance of [³H]adjudin from the systemic circulation after i.v. administration.

FIG. 11

Effects of the adjudin-FSH mutant conjugate on the testis, kidney, liver, and small intestine. Adjudin-FSH mutant conjugate (50 µg containing ~0.5 µg of adjudin/kg b.wt.) was administered to adult rats (~300 g b.wt., n = 8 per time point) intraperitoneally via a 28-gauge needle. Rats were sacrificed at 2, 4, 6, and 12 weeks thereafter for histological analysis by hematoxylin & eosin staining. A, cross-section of the testis from normal rats (control) without treatment. B, by 2 weeks after treatment, almost all elongating/elongated spermatids were depleted from the seminiferous epithelium. C, by 4 weeks, ~98% of the tubules examined were devoid of spermatids and spermatocytes, and the tubule diameter was reduced by ~35%. D, magnified view of the boxed area in C, showing a tubule that contained only Sertoli cells, spermatogonia, and a few spermatocytes. E, by 6 weeks, germ cells began to repopulate the seminiferous epithelium. F, by 12 weeks, ~90% of tubules were indistinguishable from those of the control testes. G to I, cross-sections of kidney (G), liver (H), and small intestine (I) 4 weeks after treatment. No histological changes were detected in these organs at this time or at 6 or 12 weeks after treatment versus control rats. In addition, no histological changes were detected in skeletal muscle versus control rats at 4, 6, or 12 weeks after treatment (data not shown). Bars in A to C and E to I, 150 µm; D, 40 µm. J, changes in testes weights (organ pair) after treatment. K, summary of the fertility test (n = 4 rats) results. The fertility efficacy of control rats was arbitrarily set at 100%. [Reproduced from Mruk DD, Wong CH, Silvestrini B, and Cheng CY (2006) A male contraceptive targeting germ cell adhesion. Nat Med 12:1323–1328. Copyright © 2006 Nature Publishing Group. Used with permission.]