mTORC1/rpS6 and spermatogenic function in the testis - insights from the adjudin model

Abstract

mTORC1/rpS6 signaling complex promoted Sertoli blood-testis barrier (BTB) remodeling by perturbing Sertoli cell-cell adhesion site known as the basal ectoplasmic specialization (ES). mTORC1/rpS6 complex also promoted disruption of spermatid adhesion at the Sertoli-spermatid interface called apical ES. Herein, we performed analyses using the adjudin (a potential non-hormonal male contraceptive drug) model, wherein adjudin was known to perturb apical and basal ES function at high dose. Through direct administration of adjudin to the testis, adjudin at doses that failed to perturb BTB integrity per se, overexpression of an rpS6 phosphomimetic (i.e., constitutively active) mutant (i.e., p-rpS6-MT) that modified BTB function considerably potentiated adjudin efficacy. This led to disorderly spatial expression of proteins necessary to maintain the proper cytoskeletal organization of F5-actin and microtubules (MTs) across the seminiferous epithelium, leading to germ cell exfoliation and aspermatogenesis. These findings yielded important insights regarding the role of mTORC1/rpS6 signaling complex in regulating BTB homeostasis.

Introduction

Studies have shown that homeostasis of the testis in adult rats is supported by several endogenously expressed (or produced) biomolecules in a local regulatory axis that coordinates cellular events across the epithelium of seminiferous tubules, the functional unit that produces millions of germ cells in an adult male on a daily basis, during the epithelial cycle of spermatogenesis [1, 2]. For instance, Sertoli cells in the seminiferous epithelium express the mTORC1/rpS6 (mammalian target of rapamycin complex 1/ribosomal protein S6) signaling complex, which is an endogenously expressed signaling protein complex that regulates Sertoli cell blood-testis barrier (BTB) dynamics based on studies in vitro [3–5] and also in vivo [6], besides its prominent role in modulating cellular energy status. In brief, the mTORC1/rpS6 signaling pathway (via rpS6 as the regulatory biomolecule) is effective to induce BTB remodeling, facilitating the transport of preleptotene spermatocytes across the immunological barrier at late stage VII-VIII of the epithelial cycle [7, 8], so that meiosis I/II and post-meiotic spermatid development can take place behind the BTB at the adluminal compartment [9, 10]. Furthermore, rpS6 is also involved in modulating apical ES function since its overexpression in the testis in vivo also induces germ cell exfoliation besides perturbing the Sertoli BTB function [6].

Results of our earlier findings are consistent with data obtained based on the use of genetic mouse knockout (KO) models of either mTOR (mammalian target of rapamycin) [11], or Raptor (regulatory-associated protein of mTOR, an adaptor which when binds to mTOR create the mTORC1 complex) [12]. Collectively, these genetic models support our earlier observations that the mTORC1 complex regulates BTB and germ cell adhesion through its effects on cytoskeletal organization. Based on these findings, we have since prepared a more potent form of rpS6 called p-rpS6-mutant (p-rpS6-MT) - a quadruple phosphomimetic (i.e., constitutively active) mutant of rpS6 - by converting p-rpS6-S235/S236 and p-rpS6-S240/S244 residues to E235/S236 and S240/S244 by site-directed mutagenesis [5].

Studies have shown that overexpression of this p-rpS6-MT in the testis in vivo, this was capable of inducing Sertoli cell BTB remodeling, and because of its constitutively active nature, it was also able to induce other unexpected effects on the status of spermatogenesis effectively vs. the WT (wild type rpS6 without mutations), most notably germ cell exfoliation from the testis, besides BTB remodeling [6]. On the other hand, adjudin, 1-(2,4-dichlorobenzyl)-1H-indazole-carbohydrazine, was shown to be a potent non-hormonal male contraceptive by inducing rapid germ cell exfoliation from the testis with virtually no remarkable acute toxicity [13, 14]. However, when adjudin was used at low-doses alone, it had no effects on spermatogenic function due to its relative low bioavailability [13], failing to cause any phenotypes in the testis [13, 15]. Based on these findings, we sought to investigate if the p-rpS6-MT-mediated barrier modification at the BTB would have any physiological relevance. For instance, could the use of p-rpS6-MT alter the transport function of a drug, such as adjudin, at the BTB by promoting its entry into the testis? Thus, when adjudin was administered to adult rats at low dose, transient overexpression of p-rpS6-MT that perturbed BTB function could improve the bioavailability of adjudin, thereby perturbing spermatid adhesion in the seminiferous epithelium as if a higher dose of adjudin was used. Herein, we performed a careful analysis through the combined use of adjudin, ranging from low to high doses (i.e., without and with effects on spermatogenic function by itself in the testis), and p-rpS6-MT overexpression for their local administration to the rat testis. Furthermore, this animal model was used to examine the underlying mechanism by which p-rpS6-MT induced BTB remodeling and germ cell exfoliation.

Materials and Methods

Animals.

Adult male Sprague-Dawley rats at 250–300 g b.w. (about ~60 days of age) were purchased from Charles River Laboratories (Kingston, NY). The use of animals and detailed experimental procedures including respective treatments of these rats as reported herein were approved by the Rockefeller University Institutional Animal Care and Use Committee (Protocol Numbers: 15–780-H and 18043-H). The use of toxicant cadmium chloride in this study was approved by the Rockefeller University Laboratory Safety and Environmental Health (RU LS&EH). Furthermore, the use of recombinant DNA materials including plasmid DNAs containing different cDNA constructs for animal studies was approved by the Rockefeller University Institutional Biosafety Committee (Approval Number: 2-15-04-007). At specific time points, rats were euthanized by CO₂ asphyxiation using slow (20%−30%/min) displacement of chamber air with compressed carbon dioxide using a euthanasia chamber with a built-in gas regulator approved by RU LS&EH.

Antibodies.

All antibodies used for our experiments reported herein were obtained commercially, except otherwise specified, and listed in Table 1.

Table 1.

Antibodies used for the different experiments in this report

Antibody (RRID)	Host Species	Vendor	Catalog Number	Application (working dilution)
β-actin (AB_630836)	Goat	Santa Cruz Biotechnology	sc-1616	IB (1:300)
Arp3 (AB_476749)	Mouse	Sigma-Aldrich	A5979	IF (1:200)
β-Tubulin(AB_2210370)	Rabbit	Abcam	Ab6046	IF (1:500)
EB1 (AB_2141629)	Rabbit	Santa Cruz Biotechnology	sc-15347	IF (1:300)
				IB (1:200)
Eps8 (AB_397544)	Mouse	BD Biosciences	610143	IF (1:100)
N-Cadherin (AB_2313779)	Mouse	Thermo Fisher Scientific	33-3900	IF (1:100)
MARK4(AB_2140610)	Rabbit	Protein Technology Group	20174-1-AP	IF (1:100)
Vimentin (AB_628437)	Mouse	Santa Cruz Biotechnology	sc-6260	IF (1:500)
ZO-1 (AB_2533938)	Rabbit	Thermo Fisher Scientific	61-7300	IF (1:100)
rpS6(AB_331355)	Rabbit	Cell Signaling Technology (Danvers, MA)	2217	IB (1:1000)
p-rpS6 S235/S236 (AB_916156)	Rabbit	Cell Signaling Technology	4858	IB (1:2000)
p-rpS6 S240/S244 (AB_10694233)	Rabbit	Cell Signaling Technology	5364	IB (1:1000)
GAPDH(AB_2107448)	Mouse	Abcam	Ab8245	IB (1:1000)
Goat IgG-Alexa 488 (AB_2534102)	Donkey	Thermo Fisher Scientific	A-11055	IF (1:250)
Goat IgG-Alexa 555 (AB_2535853)	Donkey	Thermo Fisher Scientific	A-21432	IF(1:250)
Mouse IgG Alexa Flour 488 (AB_2534088)	Goat	Thermo Fisher Scientific	A-11029	IF (1:250)
Mouse IgG Alexa Flour 555 (AB_2534088)	Goat	Thermo Fisher Scientific	A-21424	IF (1:250)
Rabbit IgG Alexa Flour 488 (AB_2576217)	Goat	Thermo Fisher Scientific	A-11034	IF (1:250)
Rabbit IgG Alexa Flour 555 (AB_2535850)	Goat	Thermo Fisher Scientific	A-21429	IF (1:250)

RRID, Resource Identification Initiative number; Arp3, actin-related protein 3; EB1, end binding protein 1; Eps8, epidermal growth factor receptor pathway substrate 8; MARK4, microtubule affinity-regulating kinase 4; rpS6, ribosomal protein S6; ZO-1, zonula occludens 1; IgG, immunoglobulin G.

Administration of adjudin to the testis.

Adjudin was dissolved in ethanol as a 1 mg/ml working solution, which was then administered to the testis in appropriate volume to obtain a desired level of 7.5 μg, 75 μg or 150 μg adjudin per testis as described [6] (see regimen in Figure 1).

Figure 1. Local administration of the non-hormonal male contraceptive adjudin in the testis coupled with p-rpS6-MT overexpression promotes adjudin-induced aspermatogenesis in rats.

(A) Regimen used for the experiments reported herein. This regimen was used for two independent experiments, with n = 3 rats at each termination time point for treatment vs. control groups in the first experiment, and n = 6 rats in the second experiment. (B) In testes of control (pCI-neo/Ctrl, empty vector) and adjudin low dose (7.5 μg/testis, at both 4D and 2W) group, the seminiferous epithelium was normal wherein polarized (with their heads pointed toward the basement membrane, and their tails to the tubule lumen) elongating/elongated spermatids were properly aligned across the epithelium and Sertoli cell nuclei (yellow arrowheads) located near the basement membrane. However, when adjudin was used at low dose (7.5 μg/testis) together with overexpression of p-rpS6-MT led to considerable defects in spermatogenesis, considerable more obvious than overexpression of p-rpS6-MT alone as noted herein, illustrating the possibility the p-rpS6-MT that modified the BTB function facilitated the entry of adjudin into the adluminal compartment to induce defects in spermatogenesis. Defects induced by adjudin at 75 or 150 μg/testis with p-rpS6-MT was also considerable more severe than adjudin alone at either one of these doses, further confirming the notion that p-rpS6-MT facilitated the entry of adjudin into the testis behind the BTB to exert its disruptive effects to induce spermatogenesis defects. These defects include: (i) germ cell loss, such as unwanted spermiation wherein spermatid/spermatocytes were noted in tubule lumen; (ii) retention of step 19 spermatids in the epithelium (blue arrowheads) that failed to undergo spermiation at stage VIII of the cycle due to defects in MT-based cytoskeleton to support their transport to the tubule lumen; (iii) formation of multi-nucleated cells composed of round spermatids (green arrowheads) or giant multi-nucleated (round spermatid-based) cells (white arrowheads); (iv) giant phagosomes resulting from degradation of multi-nucleated spermatid cells (red arrowheads) were found near the tubule lumen instead of in the basal region; (v) type A spermatogonial cells detached from the basement membrane (pink arrowhead); (vi) elongated spermatids with defects in polarity (some are also malformed with curved spermatid heads) in which their heads no longer pointed toward the basement membrane (black arrowheads) and (vii) considerable thinning of the seminiferous epithelium due to germ cell loss. Scale bars, 350 μm, 100 μm, and 80 μm for the 1^st, 2^nd, and 3^rd column, respectively; magnified images enlarged from 3^rd or 2^nd column encircled in green, red and yellow boxes are 60 μm, 25 μm, and 20 μm, respectively. (C) Results of immunoblot analysis from a representative experiment of n=3 independent experiments using different rat testes. About 40 μg protein from lysates of testes was used per lane, confirming overexpression of the corresponding rpS6 and its activated/phosphorylated forms. GAPDH and β-actin served as protein loading controls. (D) The parameters used to assess the % of tubules with defects in spermatogenesis as noted in (B) were shown in this histogram. In order to provide semiquantitative analyzed data regarding the extent of defects in different treatment vs. control groups, the thinning of seminiferous epithelium alone that illustrated the extent of defects in spermatogenesis was also used to assess % of tubule damage (see right panel). About 150 tubules were randomly selected and scored for defects in sections of testes in a rat, to a total of 600 tubules from 4 testes of different rats. Each bar represents a mean±SD scored tubules from n=4 rats. D, day; W, week. *, P<0.05; **, P<0.01; ***, P<0.001.; ns, non-significantly different.

Overexpression of rpS6 quadruple phosphomimetic (constitutively active) mutant (p-rpS6-MT) and treatment with or without adjudin in adult rat testes.

p-rpS6-MT was generated by site-directed mutagenesis using a full-length cDNA of rpS6 cloned from Sertoli cell total cDNAs by converting Ser(S)235, S236, S240, and S244 to Glu (E)235, E236,E240, and E244 (i.e., p-rpS6-MT) as detailed elsewhere [5]. This mutant was then cloned into the mammalian expression vector pCI-neo (Promega, Madison, WI). Prior to transfection, plasmid DNA containing with or without (i.e., pCI-neo empty vector, serving as the negative control) the p-rpS6-MT, was obtained by using Plasmid Plus Midi kits (Qiagen, Boston, MA) to eliminate possible endotoxin contamination. In vivo transfection was performed by intratesticular injection using 75 μl transfection solution per testis (note: testes also received with or without adjudin administration at 7.5, 75 or 150 μg/testis dissolved in ethanol, simultaneously but at a separate site), which contained 15 μg plasmid DNA (for both treatment groups vs. control group) together with 1.8 μl Polyplus in vivo-jetPEI® reagent (Polyplus transfection S.A., Illkirch-Graffenstaden, France) according to the manufacturer’s instructions as described [16, 17]. Transfection solution was gently administered into each testis, by inserting a 28-gauge needle with a length of 12.7mm attached to a 0.5-ml insulin syringe, from the apical to the basal region of the testis, as the syringe was withdrawn from the testis, plasmid DNA contained in the transfection medium was gently released to the testis. This thus avoided rapid buildup of hydrostatic pressure. Adjudin dissolved in ethanol in a final volume of 150 μl was gently loaded behind the tunica albuginea which was shown to rapidly diffuse into the entire testis based on pilot experiments. In short, the total volume (transfection suspension and adjudin combined) loaded into the testis and behind the tunica albuginea was at 225 μl, this thus avoided rapid build-up of hydrostatic pressure to induce unwanted epithelial damage as noted in testes of control groups when examined microscopically for histological and other analysis. At specified time, at 4D (day) and 2W (week), following transfection and/or adjudin administration, rats were euthanized by CO₂ asphyxiation. Testes were removed immediately and then either snap-frozen in liquid nitrogen (or IF, IB, Co-IP) or fixed in modified Davidson’s fixative (consisted of 30% formaldehyde (37%, w/v in H₂O), 15% ethanol, 5% glacial acetic acid, and 50% double-distilled H₂O) [18]. Testes fixed in modified Davidson’s fixative were then embedded in paraffin, and cross sections (5 μm in thickness) were obtained with a microtome. For each experiment reported herein, each time point had n=3 rats, including each experimental vs. control group. Samples from treatment and control groups with an experimental set were processed simultaneously for histological analysis, immunoblot analysis (IB), and immunofluorescence (IF) to avoid inter-experimental variations.

Estimation of transfection efficiency for overexpression of p-rpS6-MT in testes in vivo.

Transfection efficiency for overexpression of p-rpS6-MT in testes vs. pCI-neo/Ctrl (empty vector) was estimated as follows. A full-length DsRed2 cDNA obtained from pIRES2-DsRed2 (Clontech, Mountain View, CA) was cloned into pCI-neo by ligation to the MluI and XbaI sites of the pCI-neo vector [17, 19, 20]. Since this DsRed2 cDNA encoding the Discosoma sp. red fluorescence protein DsRed2, so that fluorescence aggregates of pCI-neo/DsRed2 were readily detectable by randomly scored 100 tubules from cross-sections of testis from n=3 rats on day 4 following its transfection on day 0 (15 μg pCI-neo/DsRed2 plasmid DNA per testis, similar to the other transfection groups) using the regimen noted in Figure 1A. Successful transfection referred to a seminiferous tubule cross-section with >10 aggregates of red fluorescence near the nuclei of Sertoli cells near the base of the tubule. About ~70% of the randomly scored tubules transfected with pCI-neo/DsRed2 using the Polyplus in vivo-jetPEI® transfection medium were found to be positive, illustrating a transfection efficiency of at least 70%, consistent with three earlier reports from our laboratory [17, 19, 20]. Furthermore, overexpression of rpS6 and its phosphorylated and active forms (including the phosphomimetic mutant), namely, p-rpS6-S235/S236 and p-rpS6-S240/S244 vs. their corresponding mutants, were assessed by IB to confirm there was an increase in the protein steady-state level of rpS6 and its phosphorylated/activated forms by using the corresponding specific antibodies (Table 1).

Histological analysis by hematoxylin and eosin staining.

For histology, cross-sections of testes mounted on microscopic slides were stained with H&E. In brief, paraffin wax sections were dissolved in xylene, and then rehydrated by incubating sections through xylene and decreasing strengths of ethanol (100%, 95%, 80%, 70%, 50%, and 0%, vol/vol in MilliQ water), and then Milli-Q water. After rehydration, sections were stained with Mayer’s hematoxylin, washed with tap water, and then stained with eosin (Richard-Allan Scientific, San Diego, CA). Thereafter, sections were mounted in Aqua-Poly-Mount (Polysciences, Warrington, PA) for microscopy. Defects in spermatogenesis were examined in cross-sections of testes using the following criteria: (i) germ cell loss when >15 germ cells, mostly round spermatids and spermatocytes, found in tubule lumen; (ii) retention of step 19 spermatids in non-stage VII-VIII tubules, intermingled with elongating spermatids; (iii) appearance of multi-nucleated round spermatids; (iv) phagosomes failed to be transported to the base of the tubule for degradation; (v) defects in spermatid polarity when their heads no longer pointed toward the basement membrane but deviated at least 90°–180° from their intended orientation; and (vi) thinning of the seminiferous epithelium due to germ cell loss. About 150 seminiferous tubules from crosssections of tubules were randomly selected and scored for defects in spermatogenesis in a rat testis, to a total of 600 tubules from 4 testes of different rats.

Immunofluorescence (IF) staining and filamentous (F)-actin staining.

Cross-sections of testes snap frozen in liquid nitrogen and stored at −80°C were obtained in a Microm (Model HM500M) cryostat at −22°C at 7-μm thickness. Frozen sections were air-dried and immediately fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS), permeabilized in 0.1% Triton X-100 in PBS (10mM sodium phosphate, 0.15M NaCl, pH 7.4, at 22°C), and subsequently blocked in 1% bovine serum albumin in PBS (wt/vol). Sections were then incubated with corresponding specific primary antibodies at appropriate dilution (Table 1), to be followed by Alexa Fluor–conjugated secondary antibodies (Alexa Fluor 555 for red fluorescence, Alexa Fluor 488 for green fluorescence; from Invitrogen). For F-actin staining, sections were incubated with fluorescein isothiocyanate (FITC)–conjugated phalloidin (green fluorescence; Invitrogen). To visualize cell nuclei, sections were incubated with 4′,6-diamidino-2-phenylindole (DAPI) (50 mg/mL in PBS) and then mounted in ProLong Gold Antifade mounting medium (Thermo Fisher Scientific). Fluorescence images were captured using the Nikon Eclipse 90i Fluorescence Microscope system using Nikon NIS Elements 3.2 Imaging Software package (Nikon Instruments Inc.). To assess changes in F-actin organization in the seminiferous epithelium with semi-quantitatively analyzed data, the relative distribution of F-actin or a BTB-associated protein (e.g., N-cadherin, β-catenin, occlduin, ZO-1) at a specific site, such as the basal ES/BTB, was assessed by measuring its distribution in cross-sections of testes from treatment vs. control groups. At least 200 tubules were randomly selected from a rat testis for assessment using n=3 testes from different rats. All IF images shown herein were representative findings from an experiment of n=3 independent experiments, which yielded similar results.

Immunoblot (IB) analysis.

IB analysis using lysates of testes (about 40 μg protein from each sample) was performed to confirm overexpression of rpS6 vs. its phosphorylated/activated forms using the corresponding specific antibodies (Table 1) between treatment and control groups as described [16]. Protein concentration in testis lysates was estimated using a Bio-Rad DC protein assay kit (Bio-Rad Lab, Hercules, CA) with BSA as a standard. Protein bands in blots were visualized by chemiluminescence using home-made kits [21], and images were captured using an ImageQuant LAS 4000 mini (GE Healthcare Life Sciences) Imaging system and ImageQuant softward package (Version 1.3) [21].

Statistical analysis.

Statistical analysis was performed with GraphPad Prism 6 software (GraphPad Software) using one-way analysis of variance (ANOVA) for multigroup comparisons. All experiments had at least 3–4 rats or samples for analysis. P<0.05 was considered statistically significant.

Results

Combined use of an endogenous BTB modifier rpS6 and low-dose adjudin through local administration to the testis induces rapid germ cell exfoliation and aspermatogenesis in adult male rats.

Using the regimen outlined in Figure 1A, testes of rats from control (pCI-neo/Ctrl, empty vector) or adjudin alone at 7.5 μg/testis treatment group terminated at 4D (day) or 2W (week) had no apparent effects on the status of spermatogenesis (Figure 1B). While a single transfection by overexpressing p-rpS6-MT alone in the testis had mild but notably effects on spermatogenesis by 2W (but not 4D), its combined use with adjudin at 7.5, 75 or 150 μg/testis induced wide-spread aspermatogenesis in >98% of the tubules examined in n = 9 rats (for each treatment vs. control groups) by 4D and 2W (Figure 1B). When adjudin was used alone, considerable effects were noted only in 75 and 150 μg/testis group by 2W. The defects of spermatogenesis that were noted included germ cell loss, defects in spermatid transport and defects in spermatid morphology, appearance of multi-nucleated round spermatids which would be engulfed by Sertoli cells to form phagosomes to be disposed via the intracellular lysosomal degradation pathway, and also considerable thinning of the seminiferous epithelium due to germ cell loss (Figure 1B). The transfection efficiency was estimated to be ~70% when DsRed2, a red fluorescence protein from Discosoma sp. was cloned into pCI-neo for its overexpression in the testis using the regimen in Figure 1A with rats (n=3 rats), and terminated on day 4 for examination. Positive transfection referred to a seminiferous tubule cross-section with >10 aggregates of red fluorescence near the nuclei of Sertoli cells close to the base of the tubule when transfection was performed using pCI-neo/DsRed2 plasmid DNA and Polyplus in vivo-jetPEI® transfection medium. Furthermore, overexpression of rpS6 (total) and its corresponding activated/phosphorylated forms following overexpression was confirmed (Figure 1C). Adjudin was also capable of inducing an up-regulation on the expression of p-rpS6-S235/S236 and - S240/S244, but not total rpS6, and this finding is consistent with an earlier report [3]. It was noted that when adjudin was used at a relative high dose at 150 μg/testis (and without p-rpS6-MT for overexpression), it consistently down-regulated rpS6 expression and also its two activated/phosphorylated forms (Figure 1C). However, overexpression of p-rpS6-MT remained capable of up-regulating rpS6 (total) and its two activated/phosphorylated forms (Figure 1C). In brief, these findings are significant, since they unequivocally demonstrated that overexpression of p-rpS6-MT that modified the BTB by making it “leaky” transiently potentiated the entry of adjudin into the testis to induce germ cell exfoliation, leading to infertility since >98% of the tubules were affected (Figure 1B). This possibility as noted in Figure 1B was confirmed when the % of defective tubules was analyzed in different treatment vs. control groups (Figure 1C) wherein overexpression of p-rpS6-MT considerably promoted the effectiveness of adjudin.

p-rpS6-MT, but not adjudin, effectively modifies the BTB integrity in vivo.

To better understand the effects of p-rpS6-MT vs. adjudin alone and p-rpS6-MT+adjudin on the BTB integrity in vivo, we performed a detailed analysis of the BTB function in vivo based on the regimen in Figure 1A using n=4 rats for each treatment and control groups for assessing BTB integrity as shown in Figure 2. In testes of control (pCI-neo/Ctrl) rats, the presence of a functional BTB was capable of preventing the entry of a small membrane impermeable sulfo-NHS-LC-biotin (Mr 556.59) into the adluminal compartment. This is in sharp contrast to rats treated with CdCl₂ (3 mg/kg b.w., by i.p.) which was shown to induce irreversible BTB disruption [22–24], wherein biotin rapidly entered the adluminal compartment to biotinylate proteins across the entire seminiferous epithelium of any tubule examined (Figure 2). Consistent with earlier reports [25], adjudin when used at 75 or 150 μg/testis at 4D and 2W, the BTB remained intact (Figure 2). It was of interest to note that the use of p-rpS6-MT alone was capable of modifying the BTB integrity, making it “leaky” by 4D, but the BTB was capable of being “resealed” when a single transfection of p-rpS6-MT for its overexpression was used as noted herein (Figure 2), illustrating the effects of p-rpS6-MT on BTB function is reversible. Furthermore, the use of adjudin at 7.5 μg/testis combined with p-rpS6-MT overexpression modified the BTB by making it “leaky” at 4D, and the disrupted BTB was also “resealed” by 2W, confirming the effect of p-rpS6-MT that perturbed BTB function was reversible (Figure 2). However, the use of adjudin at 75 or 150 μg/testis plus p-rpS6-MT made the BTB “leaky” even after 2W (Figure 2), illustrating that while adjudin per se at the doses used herein was not capable of inducing BTB disruption, the combined use of p-rpS6-MT and adjudin was a potent drug formulation to modify the BTB to improve the access of adjudin to induce germ cell exfoliation and to potentiate BTB disruption. These findings also support the notion that this is an effective approach to induce aspermatogenesis and infertility in male rats, applicable to male pets, avoiding the use of tedious and expensive surgical procedures of neutering.

Figure 2. p-rpS6-MT overexpression in the testis transiently modifies the BTB in the testis in vivo, promoting the entry of adjudin into the adluminal compartment to induce germ cell exfoliation.

The regimen used for this study was shown in Figure 1A. In testes from rats transfected with pCI-neo (empty vector, pCI-neo/Ctrl), the functional BTB blocked the entry of biotin (EZ-link sulfo-NHS-LC-biotin, a membrane impermeable biotinylation reagent of Mr 556.59) into the adluminal compartment, behind the immunological barrier near the basement membrane (annotated by dashed white circle). Biotinylated proteins was visualized by Alexa Fluor 555-streptavidin (red fluorescence) that had high affinity for biotin. Thus, red fluorescence (biotin) was limited to the base of the seminiferous tubule and the interstitial space. This was in sharp contrast to the positive control in which rats were treated with CdCl₂ (3 mg/kg b.w., by i.p.; known to induce BTB disruption [24, 58]) for 3 day (D) wherein biotin entered the adluminal compartment, virtually stained the entire tubule. On the other hand, adjudin at 75 or 150 μg/testis failed to induce BTB disruption, consistent with earlier reports [25, 59, 60], confirming the notion that adjudin per se is not a BTB modifier nor a BTB disruptor, unless acute doses of adjudin were used (e.g., at 150 or 250 mg/kg b.w., by oral gavage) [25]. While overexpression of p-rpS6-MT in the testis was capable of modifying the BTB permeability by making the barrier “leaky” by day 4, its effect was transient since the barrier was “resealed” by 2-week (W), consistent with earlier reports [3, 5]. However, overexpression of p-rpS6-MT to be followed by adjudin at either 7.5, 75 or 150 μg/testis, they were able to modify BTB to make it “leaky” by 4D. At p-rpS6-MT+7.5 μg adjudin/ml testis, the BTB was able to “reseal” by 2W, but not when adjudin was used at a dose of either 75 or 150 μg/testis, illustrating the combined use of p-rpS6-MT and adjudin at these doses were able to make the barrier “leaky” for an extended period. Scar bars, 450 μm and 100 μm, in inset and enlarged image, respectively, which apply to corresponding insets and micrographs. D, day; W, week.

Local p-rpS6-MT overexpression in the testis coupled with adjudin administration that induces aspermatogenesis is mediated through MT disorganization, via changes in the spatial expression of MT regulatory proteins.

Changes in MT organization.

MT organization in the seminiferous epithelium was visualized and assessed by immunostaining of α-tubulin (green fluorescence), which together with β-tubulin create the α-/β-tubulin heterodimers that serve as the building blocks of MTs [26, 27]. In testes of control or low-dose adjudin (7.5 μg/testis) treated groups (n = 4 rats), MTs appeared as tracks that laid perpendicular to the basement membrane (annotated by dashed white line) and stretched across the entire seminiferous epithelium to support transport of spermatids and other organelles (e.g., residual bodies, phagosomes, mitochondria, endocytic vesicles) [28, 29]. However, following local overexpression of p-rpS6-MT in the testis, it also induced gross disorganization of MTs in the seminiferous epithelium by 2W (but not at 4D), or local administration of adjudin at 75 or 150 μg/testis at 4D and 2W (Figure 3). However, combined transfection of p-rpS6-MT for its overexpression and adjudin treatment at all three doses at 7.5, 75 and 150 μg/testis, MT organization was perturbed at 4D and at 2W (Figure 3). These findings thus support the notion that the combined overexpression of p-rpS6-MT that modified the BTB to facilitate the entry of adjudin, even at 7.5 or 75 μg/testis for 4D, was potent and effective enough to induce aspermatogenesis, causing sterility, making this procedure suitable to replace surgical procedures of neutering pets.

Figure 3. p-rpS6-MT overexpression in the testis that transiently modifies the BTB function promotes adjudin-induced disruptive changes on MT-based cytoskeletal organization.

In testes of control (pCI-neo/Ctrl) and low dose adjudin (7.5 μg/testis) groups, MTs (green fluorescence, visualized by α-tubulin staining since α-tubulin together with β-tubulin create the α-/β-tubulin heterodimers which are the building blocks of MTs) appeared as tracks that laid perpendicular to the basement membrane (annotated by dashed white line) and stretched across the entire seminiferous epithelium by 4D (day) and 2W (week) following transfection with empty vector (Ctrl) or adjudin treatment. While p-rpS6-MT transfection alone also induced defects in MT organization across the epithelium by 2W (but not 4D), combined adjudin treatment (even at 7.5 μg/testis vs. 75 or 150 μg/testis) + p-rpS6 overexpression was considerably more effective to induce MT disorganization (and also better than this dose adjudin alone), wherein the MT-based tracks were grossly truncated, mis-aligned (i.e., no longer laid perpendicular but parallel to the basement membrane) and even collapsed and laid flat near the basement membrane (annotated by dashed white line) when adjudin was used at 75 or 150 μg/testis by 2W combined with p-rpS6-MT overexpression. Insets in red were magnified images also boxed in red in the corresponding lower magnified image. Scale bar, 120 μm, inset at 80 μm, inset.

Changes in spatial expression of MT regulatory proteins EB1 and MARK4.

We next sought to examine the mechanism by which p-rpS6-MT and low-dose adjudin that perturb MT organization. EB1 (end binding protein 1) is a plus (+)-end tracking protein (+TIP) known to induce MT stability [30, 31], whereas MARK4 (microtubule affinity-regulating kinase 4) is a Ser/Thr protein kinase that its phosphorylation on MAPs (microtubule-associated proteins, such as MAP1a) induces detachment of MAPs from MTs, thereby leading to MT destabilization and MT catastrophe [32, 33]. Both EB1 [34] and MARK4 [35] have also been studied in the testis. In testes of control (pCI-neo/Ctrl) or low-dose adjudin (7.5 μg/testis) treated groups, EB1 (red fluorescence) (Figure 4) or MARK4 (red fluorescence) (Figure 5) co-localized with MTs (green fluorescence) and appeared as tracks that laid perpendicular to the basement membrane that stretched across the entire seminiferous epithelium. However, the pattern of distribution and expression of either EB1 (Figure 4) or MARK4 (Figure 5) were virtually identical to changes in the MT organization following high-dose adjudin (75 or 150 μg/testis) treatment or p-rpS6-MT overexpression alone as noted in Figure 3. Collectively, the findings reported in Figures 3–5 thus support the notion that the disruptive changes in MT organization that was considerably more severe as noted in the testis p-rpS6-MT+adjudin (at 7.5, 75 or 150 μg/testis) group was mediated by changes in the spatiotemporal expression of EB1 and MARK4.

Figure 4. Combined use of overexpression of p-rpS6-MT and low-dose adjudin that induces MT cytoskeletal disruption in the testis is mediated through changes in the spatiotemporal expression of MT regulatory protein EB1.

In testes of control and low-dose adjudin (7.5 μg/testis) alone groups, EB1 (red fluorescence, a +TIP known to stabilize MTs in mammalian cells/epithelia [31]) that stretched across the entire seminiferous epithelium and laid perpendicular to the basement membrane was found to co-localize with MTs (green fluorescence, visualized by α-tubulin staining) as earlier reported [34]. While overexpression of p-rpS6-MT alone was capable of inducing some disruptive changes on the spatiotemporal expression of EB1 that affected MT organization across the epithelium by 2W (week), but not by 4D (day), its overexpression combined with adjudin at 7.5 μg/testis was able to induce notable changes in EB1 spatiotemporal expression and MT organization at both 4D and 2W, in sharp contrast to adjudin used at 7.5 μg/testis alone. Furthermore, these disruptive changes were considerably more remarkable with adjudin was used at 75 and 150 μg/testis together with p-rpS6-MT overexpression at 4D and 2W. Yet adjudin used at 75 μg/testis alone also had mild effects on both MARK4 and MT distribution/organization. But adjudin alone at high dose (150 μg/testis) was capable of inducing disruptive distribution and organization of MARK4 and MT, respectively. Collectively, these findings supported the notion that p-rpS6-MT modified the BTB function, making it “leaky”, thereby facilitating the entry of adjudin into adluminal compartment to exert is disruptive effects on spermatogenesis. Insets in red were magnified images also boxed in red in the corresponding lower magnified image. Scale bar, 120 μm, inset at 80 μm, inset.

Figure 5. Combined use of overexpression of p-rpS6-MT and low-dose adjudin that induces MT cytoskeletal disruption in the testis is mediated through changes in the spatiotemporal expression of MT regulator protein MARK4.

In testes of control and low-dose adjudin (7.5 μg/testis) alone groups, MARK4 (red fluorescence, a Ser/Thr protein kinase that phosphorylates MAPs, causing their detachment from MTs, destabilizing MT that leads to MT catastrophe [32, 61] that stretched across the seminiferous epithelium and laid perpendicular to the basement membrane was found to co-localize with MTs (green fluorescence) as earlier reported [35]. While overexpression of p-rpS6-MT alone was capable of inducing some disruptive changes on the spatiotemporal expression of MARK4 and MT organization across the epithelium by 2W (week), but not by 4D (day), its overexpression combined with adjudin at 7.5 μg/tesis was able to induce notable changes in EB1 localization and MT organization at both 4D and 2W, in sharp contrast to adjudin used at 7.5 μg/testis alone. Furthermore, these disruptive changes were more remarkable when adjudin was used at 75 and 150 μg/testis at both 4D and 2W. Yet adjudin used at 75 μg/testis alone also had mild effects on both MARK4 and MT distribution/organization. But adjudin alone at high dose (150 μg/testis) was capable of inducing disruptive distribution and organization of MARK4 and MT, respectively. Collectively, these findings supported the notion that p-rpS6-MT modified the BTB function, making it “leaky”, thereby facilitating the entry of adjudin into the adluminal compartment to exert its disruptive effects on spermatogenesis. Insets in red were magnified images also boxed in red in the corresponding lower magnified image. Scale bar, 120 μm, inset at 80 μm, inset.

Local overexpression of p-rpS6-MT coupled with adjudin administration in the testis that induces aspermatogenesis is mediated through cytoskeletal disorganization of actin, via changes in the spatial expression of actin regulatory proteins.

Changes in F-actin organization.

Studies have shown that the actin-based cytoskeleton in the testis across the seminiferous epithelium, in coordination with the MT-based cytoskeleton, is crucial to support spermatogenesis via their cycles of assembly and disassembly to confer plasticity to support the transport of spermatids and other organelles (e.g., residual bodies, phagosomes, mitochondria) [36–38] during the epithelial cycle of spermatogenesis. Furthermore, F-actin is the core structural component of the apical and basal ES to support spermatid and Sertoli cell adhesion, cell polarity, and spermatogenesis [39–41]. As noted herein, in testes of control (pCI-neo/Ctrl) or low-dose adjudin only (7.5 μg/testis) treated rats, F-actin was prominently expressed and localized at the apical ES, alongside with elongating/elongated spermatids and also at the basal ES/BTB near the basement membrane (annotated by dashed white line) at 4D and 2W (Figure 6). Similar to MTs, while p-rpS6-MT overexpression was capable of inducing F-actin disorganization by 2W (but not at 4D), combined use of p-rpS6-MT overexpression and adjudin (even at low-dose of 7.5 μg/testis) was considerably more effective to induce F-actin disorganization than adjudin alone at either 75 or 150 μg/testis (Figure 6).

Figure 6. p-rpS6-MT overexpression in the testis that transiently modifies the BTB function promotes adjudin-induced disruption on actin-based cytoskeletal organization.

In testes of control (pCI-neo/Ctrl) and low dose adjudin alone (7.5 μg/testis, without p-rpS6-MT overexpression) groups, MTs (green fluorescence, visualized by α-tubulin staining since α-tubulin together with β-tubulin create the α-/β-tubulin heterodimers which are the building blocks of MTs) appeared as tracks that laid perpendicular to the basement membrane (annotated by dashed white line) and stretched across the entire seminiferous epithelium by 4D (day) and 2W (week) following transfection with empty vector (Ctrl) or adjudin treatment. While p-rpS6-MT transfection alone also induced defects in MT organization across the epithelium by 2W (but not 4D), combined adjudin treatment (even at 7.5 μg/testis vs. 75 or 150 μg/testis) + p-rpS6-MT overexpression was considerably more effective to induce MT disorganization (and also better than this dose adjudin alone), wherein the MT-based tracks were grossly truncated, mis-aligned (i.e., no longer laid perpendicular but parallel to the basement membrane) and even collapsed and laid flat near the basement membrane (annotated by dashed white line) when adjudin was used at 75 or 150 μg/testis by 2W combined with p-rpS6-MT overexpression. Insets in red were magnified images also boxed in red in the corresponding lower magnified image. Scale bar, 120 μm, inset at 80 μm, inset.

Changes in distribution of adhesion proteins at the BTB.

It is noted that N-cadherin (an integral membrane protein at the basal ES, green fluorescence) (Figure 7, upper panel) and ZO-1 (an adaptor protein at the TJ, green fluorescence) (Figure 7, lower panel) are BTB-associated proteins using F-actin for their attachment. We thus sought to examine any changes on their distribution following different treatment groups compared to controls (Figure 7). In testes of control (pCI-neo/Ctrl) or low-dose adjudin only (7.5 μg/testis) treated rats at both time points, both BTB-associated proteins were tightly localized to the BTB near the basement membrane (annotated by dashed white line) (see white brackets) (Figure 7). While p-rpS6-MT overexpression alone was able to induce changes in their distribution since these proteins diffused away from the BTB site (see yellow brackets vs. white brackets in control and adjudin alone low-dose groups), the presence of low-dose adjudin considerably further perturbed their distribution (Figure 7). These data were also semi-quantitatively compared between different groups (see bar graphs on right panel) (Figure 7), supporting the notion that overexpression of p-rpS6-MT considerably potentiated the disruptive effects of adjudin to induce changes in distribution of N-cadherin and ZO-1 at the BTB site. In summary, these finding thus supported the notion that overexpression of p-rpS6-MT potentiated the disruptive effects of adjudin alone on the distribution of these two basal ES- and TJ-associated proteins at the BTB.

Figure 7. p-rpS6-MT overexpression in the testis that transiently modifies the BTB function perturbs distribution of basal ES and TJ proteins at the BTB.

In testes of control (pCI-neo/Ctrl) and low-dose adjudin alone (7.5 μg/testis) groups, basal ES protein N-cadherin (an integral membrane protein) (upper panel) and TJ adaptor protein ZO-1 (a cytosolic protein that serves as an adaptor for integral membrane proteins occludin, CAR, JAM-A and others) (low panel) localized tightly to the BTB located adjacent to the basement membrane (annotated by a dashed white line) (see white brackets) at both 4D and 2W time points. While p-rpS6-MT transfection alone also induced changes in the distribution of these two BTB-associated proteins at the site by making them diffusely localized (see yellow brackets), combined adjudin treatment (even at 7.5 μg/testis vs. 75 or 150 μg/testis) plus p-rpS6-MT overexpression was considerably more effective to induce re-distribution of N-cadherin and ZO-1 at the site (see yellow vs. white brackets). Scale bar, 40 μm, which applies to other images in the corresponding panels.

Changes in spatial expression of actin regulatory proteins Arp3 and Eps8.

Arp3 (actin-related protein 3) creates a protein complex with Arp2 known as the Arp2/3 complex, which when activated by N-WASP (neuronal Wiskott-Aldrich syndrome protein), the protein complex is capable of inducing branched actin polymerization, effectively converting bundled actin filaments at the ES (e.g., basal ES at the BTB) into a branched network [42, 43]. On the other hand, Eps8 (epidermal growth factor receptor pathway substrate 8) is an actin barbed end capping and bundling protein, capable of bundling actin filaments into bundles, such as those found at the ES, to maintain ES integrity [44–46]. Thus, the combined actions of Arp2/3 and Eps8 confer plasticity to the ES in the testis, such as at the basal ES to support BTB homeostasis during the epithelial cycle of spermatogenesis. As noted in Figure 8, Arp3 and Eps8 appeared as prominent bulb-like structures, localized at the concave (ventral) side of spermatid (see also insets) at the apical ES, but also at the basal ES/BTB (see yellow arrowheads) near the basement membrane (annotated by the dashed white line) in control testes, but also in testes from rats treated with low-dose adjudin alone (7.5 μg/testis). While adjudin at 75 and 150 μg/testis was capable of inducing changes in the distribution of these two actin regulatory proteins, combined with overexpression of p-rpS6-MT was more potent and effective to induce disruptive changes in their distribution, better than p-rpS6-MT overexpression alone (Figure 8). These findings thus support the notion that changes in F-actin distribution across the seminiferous epithelium as noted in Figure 6, which in turn perturbed distribution of basal ES- and TJ-proteins at the BTB (Figure 7), was the result of changes in spatial expression of the two actin regulatory proteins Arp3 and Eps8 (Figure 8).

Figure 8. p-rpS6-MT overexpression in the testis promotes adjudin-induced disruptive changes on the spatial expression of actin regulatory proteins Arp3 and Eps8.

In control testes and also testes treated with adjudin at 7.5 μg/testis alone, Arp3 (capable of inducing branched actin polymerization, converting bundled actin filaments into a branched network) and Eps8 (an actin barbed end capping and bundling proteins that assembles actin filaments into bundles to confer ES integrity) appeared as bulb-like structures at the concave (ventral) side of spermatid heads at the apical ES. Arp3 and Eps8 were also expressed at the base of the tubule, near the basement membrane (annotated by dashed white line), at the site of the BTB (annotated by yellow arrowheads). The spatial expression of these two actin regulatory proteins was necessary to confer ES integrity to support spermatogenesis. However, adjudin alone at 150 μg/testis (but not 75 μg/testis) was found to perturb the spatial expression of these two regulatory proteins. However, overexpression of p-rpS6-MT alone by 2W also perturbed the spatial distribution of Arp3 and Eps8 (but not 4D), but overexpression of p-rpS6-MT together with adjudin at 7.5 μg/testis (at 2W) or at 75 and 150 μg/testis (at both 4D and 2W) were able to induce extensive disruptive distribution of these two actin regulatory proteins. Scale bar, 40 μm and 20 μm in inset, which apply to corresponding micrographs and insets.

Discussion

mTOR is a well conserved non-receptor Ser/Thr protein kinase which was first discovered in yeasts through the use of the antibiotic rapamycin shown to inhibit its intrinsic kinase activity [47, 48]. Subsequent studies have shown that the binding of mTOR to its adaptor protein Raptor (regulatory-associated protein of mTOR) or Rictor (rapamycin-insensitive companion of mTOR), generates the mTORC1 (mammalian target of rapamycin complex 1) or mTORC2 signaling complex, respectively [48–50]. Additionally, studies have shown that these signaling complexes are present in virtually mammalian cells, and are crucial regulators of cellular energy status and an array of cellular functions, and also the targets in treating different diseases [48–52]. Studies from our laboratory in recent years have shown that the mTORC1/rpS6 signaling complex plays a crucial role in regulating BTB dynamics. For instance, an activation of p-rpS6, such as through the overexpression of a phosphomimetic (i.e., constitutively active) mutant p-rpS6-MT in either Sertoli cell epithelium in vitro or the testis in vivo, led to a disruption of the Sertoli BTB integrity, making the barrier leaky in vitro [4, 5, 53] and also in vivo [6]. Interestingly, an 80 kDa fragment derived from the C-terminal region of laminin-a2 chain in the basement membrane that was shown to modulate BTB function also exerted its effects through the mTORC1/p-rpS6 signaling complex, involving a considerable surge in expression of p-rpS6-S235/S236 and p-rpS6-S240/S244 and a concomitant downregulation of the Akt1/2 signaling protein downstream [54, 55]. In brief, a knockdown of laminin-α2 chain by RNAi that perturbed the Sertoli cell BTB function was associated with an up-regulation on the expression of p-rpS6-S235/S236 and p-rpS6-S240/S244 and a down-regulation of Akt1/2 [55], consistent with earlier reports regarding the mTORC1/rpS6 signaling complex on BTB dynamics [3–5]. These findings that illustrate the BTB promoting effects of the 80 kDa laminin because its knockdown by RNAi perturbs the tight junction barrier function are physiologically important. As these findings demonstrate that this 80 kDa laminin-α2 fragment is physiologically linked to the mTORC1/p-rpS6 signaling complex by serving the downstream signaling pathway of the 80 kDa laminin fragment.

Herein, we have demonstrated unequivocally that the p-rpS6-MT-induced BTB remodeling as detected through the use of a biotin-functional assay that tracked changes in the barrier permeability is indeed physiologically important. First, p-rpS6 is capable of modifying the transport function at the BTB, and this is supported by considerably improving the permeability of the non-hormonal male contraceptive drug adjudin [1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide, Mr 335.19] as a study model. Thus, the p-rpS6-MT-induced BTB remodeling facilitates the transport function at the immunological barrier. For instance, when adjudin was used at a dose of 75 μg/testis or 150 μg/testis, it failed to induce Sertoli cell BTB integrity in vivo as reported herein, also consistent with an earlier report that adjudin when used at low doses, even capable of inducing transient male infertility, it failed to cause the BTB to become “leaky” unless a higher dose was used [25]. Furthermore, while overexpression of p-rpS6-MT in the testis alone was able to induce BTB disruption on day 4, this disruptive effect was only transient since the BTB was “resealed” by 2-wk. However, overexpression of p-rpS6-MT together with adjudin (at 75 μg/testis or 150 μg/testis), the Sertoli cell BTB integrity was compromised by 4-day and 2-wk, confirming the notion that p-rpS6-MT is capable of inducing changes in BTB transport function. This thus facilitates the entry of adjudin into the testis so that even though adjudin was administered at a relatively low dose, the unexpected “opening” of the BTB improves its bioavailability considerably, potentiating the disruptive effects of adjudin since acute doses of adjudin were able to induce BTB disruption [25]. In this context, it is of interest to note that fewer than 5% of the adjudin administered by oral gavage is absorbed at the gastrointestinal tract [56], illustrating its poor bioavailability. However, overexpression of p-rpS6-MT alters the transport function at the BTB, such that a rather low dose of adjudin (e.g., at 75 and 150 μg/gm testis) is able to alter the phenotypes in the testis due to its considerable improvement in bioavailability.

Using this animal model, we also provide compelling evidence that p-rpS6-MT exerts its biological effects through changes in the cytoskeletal organization of MTs and F-actin across the seminiferous epithelium. These disruptive changes are mediated through an alteration on the spatial expression of the corresponding MT and F-actin regulatory proteins. For MT organization, either overexpression of p-rpS6-MT alone (or with adjudin) was able to perturb spatial expression of the +TIP (microtubule plus (+) end tracking protein) EB1 (end binding protein 1) known to promote MT stabilization [30, 31, 57]; and also MARK4 which is known to phosphorylate MAPs (microtubule affinity proteins, such as MAP-1a), causing their release from MTs, thereby destabilizing MTs that lead to catastrophe [32]. Their concerted efforts are necessary to maintain MT dynamics to support spermatogenic function [28, 29, 38]. These disruptive changes in spatial expression thus perturb homeostasis of the MT-based cytoskeletal network to support spermatogenic function, leading to aspermatogenesis as noted herein when compared to the use of p-rpS6-MT or adjudin alone. For F-actin organization, disruptive changes in the spatial expression of the Arp3 (which together with Arp2 that create the Arp2/3 protein complex is necessary to induce branched actin polymerization [42], causing de-bundling of the actin filaments at the ES [43]) and the actin barbed end capping and bundling protein Eps8 (by conferring actin their bundled configuration at the ES) also perturb F-actin network across the seminiferous epithelium. This is because the homeostasis of the F-actin network at the ES also requires the concerted efforts of the bundling proteins (e.g., Eps8) and branched actin polymerization proteins (e.g., the Arp2/3 complex) to confer F-actin its plasticity to support spermatogenesis. Collectively, these changes thus perturb the proper distribution of BTB-associated proteins, such as N-cadherin and ZO-1, to support BTB function in the testis as noted herein, contributing to BTB remodeling by altering its transport function.

In summary, rpS6, the downstream signaling protein of mTORC1, is a BTB modifier endogenously produced in the testis whereas adjudin is a non-hormonal male contraceptive known to induce germ cell exfoliation but its antifertility efficacy requires several oral doses of 50 mg/kg b.w. for administration. Herein, we had used the transient BTB modifier p-rpS6-MT, which was more potent to induce transient BTB “opening” [4–6] for its overexpression in combination with a low-dose adjudin, but administered locally to the testis, to support the concept that this approach is capable of inducing aspermatogenesis. This approach thus could replace the tedious, expensive and medically invasive surgical procedure of neutering male pets by using the laboratory male rats as a model in this proof-of-concept study. We have demonstrated that the rpS6-induced BTB remodeling indeed modifies the transport function of the immunological barrier using the adjudin model. Furthermore, we also provide insightful information that rpS6 exerts its regulatory effects on spermatogenic function through changes in the cytoskeletal organization of MTs and F-actin.

Highlights.

• mTORC1/rpS6 signaling complex is a blood-testis barrier (BTB) modifier.

• Overexpression of a quadruple phosphomimetic (i.e., constitutively active) mutant, p-rpS6-MT, can transiently modify the BTB transport function.

• Adjudin, a non-hormonal male contraceptive, at low-dose was ineffective to perturb spermatogenic function.

• Combined use of p-rpS6 and adjudin was found to modify the BTB function, thereby enhancing the anti-spermatogenic effects of adjudin.

• These findings illustrate the use of a BTB modifier can enhance the disruptive effects of drugs (and also toxicants) in the testis.