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Keywords:

  • adjudin;
  • blood–testis barrier;
  • seminiferous epithelial cycle;
  • spermatogonia;
  • spermatogonial stem cells;
  • testis

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

The blood–testis barrier (BTB) is a unique ultrastructure in the testis, which creates a specialized microenvironment in the seminiferous epithelium known as the apical (or adluminal) compartment for post-meiotic germ-cell development and for maintenance of an immunological barrier. In this study, we have demonstrated unequivocally that a functional and intact BTB is crucial for the initiation of spermatogenesis, in particular, the differentiation of spermatogonial stem cells (SSCs). It was shown that adult rats (∼300 g body weight, b.w.) treated with adjudin at 50 (low-dose) or 250 (high-dose) mg/kg b.w. by gavage led to germ-cell depletion from the seminiferous tubules and that >98% of the tubules were devoid of germ cells by ∼2 week and rats became infertile in both groups after the sperm reserve in the epididymis was exhausted. While the population of SSC/spermatogonia in the seminiferous tubules from both groups was similar to that of normal rats, only rats from the low-dose group were capable of re-initiating spermatogenesis; and by 20 weeks, greater than 75% of the tubules displayed normal spermatogenesis and the fertility of these rats rebounded. Detailed analysis by dual-labelled immunofluorescence analysis and a functional BTB integrity assay revealed that in both treatment groups, the BTB was disrupted from week 6 to week 12. However, the disrupted BTB ‘resealed’ in the low-dose group, but not in the high-dose group. Our findings illustrate that SSC/spermatogonia failed to differentiate into spermatocytes beyond Aaligned spermatogonia in the high-dose group with a disrupted BTB. In short, these findings illustrate the critical significance of the BTB for re-initiation of spermatogenesis besides SSC and spermatogonia.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Spermatogonial stem cells (SSCs) are a subpopulation (∼10%) of Asingle (As) spermatogonia, which can be identified based on transplantation analysis (Nakagawa et al., 2007). During spermatogenesis, As spermatogonia form pairs of cells called Apaired (Apr)-spermatogonia and then chains of cells up to 16 cells known as Aaligned (Aal)-spermatogonia, which are connected by intercellular bridges and synchronized with the spermatogenic wave (de Rooij & Russell, 2000). It is noted that As, Apr and Aal spermatogonia are called undifferentiated spermatogonia. The Aal spermatogonia then further divide and differentiate to form type B spermatogonia and eventually primary spermatocytes, which enter meiotic prophase (de Rooij & Russell, 2000). In rodents, one SSC (diploid, 2n) undergoes 10 mitotic divisions before entering meiotic prophase to be followed by two meiotic divisions to give rise to 4096 spermatids (haploid, 1n) theoretically (Ehmcke et al., 2006). However, more than 75% of the spermatids undergo apoptosis to avoid overwhelming the fixed number of Sertoli cells in the seminiferous epithelium in rodent testes (Cheng & Mruk, 2010). In short, even though SSCs account for <0.03% of all the germ cells in the mammalian testes (Tegelenbosch & de Rooij, 1993), they are capable of sustaining the production of >100 million spermatozoa each day in a typical man from puberty (at ∼11–14 years of age) through the entire adulthood via self-renewal, mitosis and meiosis during spermatogenesis. Furthermore, Apr spermatogonia were shown to be capable of switching back to become ‘true’ SSC via de-differentiation (Brawley & Matunis, 2004; Nakagawa et al., 2007). Nonetheless, it is expected that if SSCs are all depleted, spermatogenesis would be halted, leading to azoospermia and infertility. Indeed, previous studies have shown that the duration of infertility after exposure to irradiation and chemotherapy in humans and rodents correlated with the survival rate of SSCs (Meistrich, 1986). However, despite the importance of preservation of SSCs, these progenitor cells alone are not sufficient to maintain spermatogenesis. This was demonstrated in earlier studies in which prolonged azoospermia were found in animals exposed to toxicants even though type A spermatogonia in the seminiferous tubules persisted in treated animals (Boekelheide & Hall, 1991; Meistrich et al., 1999). It remained unclear why the existing spermatogonia in the seminiferous epithelium failed to re-initiate spermatogenesis and repopulate the tubules in the testes of these rats.

It is known that SSCs and As, Apr and Aal spermatogonia are found in areas of the seminiferous epithelium that border the interstitial tissue known as the spermatogonial stem-cell niche (de Rooij, 2009), which are located next to but ‘outside’ the BTB, nearing the interstitium where microvessels are present. In rats, leptotene spermatocytes are known to give rise to zygotene and pachytene spermatocytes that enter meiotic prophase by age 18 day post-partum when the BTB is established (Clermont & Perry, 1957), illustrating the significance of the BTB in spermatogenesis. This possibility is further supported by an earlier study in which treatment of neonatal rats with diethylstilbestrol (DES) that delayed the formation of BTB in rats also delayed differentiation of spermatogonia and thus meiosis (Toyama et al., 2001). Earlier studies from our laboratory have shown that adjudin [1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide, C15H12Cl2N4O, Mr 335.18, formerly called AF-2364] is a potential male contraceptive drug that exerts its effects by perturbing germ-cell adhesion in the seminiferous epithelium, most notably at the apical ectoplasmic specialization [apical ES, a testis-specific adherens junction type (Wong et al., 2008)]. Furthermore, it was shown that when two doses of adjudin at 50 mg/kg b.w. were administered by gavage, transient infertility was induced in rats as fertility rebounded in all the treated rats (Cheng et al., 2005; Mok et al., 2011). However, it was also noted that in some rats treated with multiple doses of adjudin at 37.5–50 mg/kg b.w. (by gavage), fertility did not rebound (Cheng et al., 2005; Mok et al., 2011). These findings thus prompted us to speculate that the adhesion of SSC and/or spermatogonia (and perhaps the BTB integrity) might have been disrupted in rats exposed to multiple doses of adjudin causing the loss of all SSC and/or spermatogonia from the tubules, which in turn led to the failure of their fertility to rebound. Herein, we sought to examine the above speculations with high doses of adjudin vs. a low-dose and control rats. Also, if the BTB was indeed disrupted by adjudin, it could be able to re-establish its functionality over time. These are the subjects of this present report.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Animals and antibodies

Sprague-Dawley (outbred) rats were purchased from Charles River Laboratories (Kingston, NY, USA). All animals were housed at the Rockefeller University Comparative Bioscience Center (CBC) with two rats per cage. Each rat had free access to rat chow and water ad libitum under controlled temperature (22 °C) and constant light–dark cycles (12 h of light and 12 h of darkness). The Rockefeller University laboratory animal facilities have been fully accredited by the American Association for Accreditation of Laboratory Animal Care. These animals were maintained in accordance with the applicable portions of the Animal Welfare Act and the guidelines in the Department of Health and Human Services publication ‘Guide for the Care and Use of Laboratory Animals’. The use of Sprague-Dawley rats in this report was approved by the Rockefeller University Institutional Animal Care and Use Committee with Protocol Numbers 06018 and 09016. Antibodies were obtained commercially from various vendors with their working dilutions for different experiments listed in Table 1.

Table 1.   Antibodies used for different experiments in this report
AntibodyVendorCatalog no.ApplicationsWorking dilution for IB or (IF)
  1. Antibodies used in this study cross-reacted with the corresponding rat proteins as indicated by the manufacturers. IB, immunoblotting; IF, immunofluorescence microscopy.

Mouse anti-PlzfCalbiochem (San Diego, CA, USA)OP128IF(1 : 125)
Rabbit anti-PlzfSanta Cruz Biotechnology (Santa Cruz, CA, USA)sc-22839IB1 : 1000
Rabbit anti-Utf1Chemicon/Millipore (Billerica, MA, USA)AB3383IB, IF1 : 1000, (1 : 800)
Goat anti-Oct4Santa Cruz Biotechnologysc-8629IB1 : 1000
Mouse anti-Thy-1Abcam (Cambridge, MA, USA)ab225IB1 : 1000
Rabbit anti-claudin-11Invitrogen (Carlsbad, CA, USA)36-4500IB, IF1 : 125, (1 : 100)
Rabbit anti-occludinInvitrogen71-1500IB, IF1 : 125, (1 : 100)
Rabbit anti-N-cadherinSanta Cruz Biotechnologysc-7939IB1 : 200
Rabbit anti-β1 integrinMillipore (Billerica, MA, USA)AMB1922ZIB1 : 1000
Rabbit anti-JAM-CInvitrogen40-9000IB1 : 150
Mouse anti-ZO-1Invitrogen33-9188IF(1 : 100)
Goat anti-actinSanta Cruz Biotechnologysc-1616IB1 : 300

Administration of adjudin to adult rats to induce infertility by depleting germ cells from the seminiferous epithelium

A suspension of 20 mg/mL of adjudin was prepared in 0.25% (wt/vol, in Milli-Q water using a Millipore Advantage A10 Water System, Millipore, Billerica, MA, USA) methylcellulose (Sigma-Aldrich, St. Louis, MO, USA) as described (Cheng et al., 2005). A total of three treatment regimens were used in this study: adult rats (∼300 g body weight, b.w.) rats received a single dose of adjudin at 50 (low-dose), 125 or 250 (high-dose) mg/kg b.w. via gavage at time 0 as described (Cheng et al., 2001) (see Table 2). A vehicle control group was also included in which rats received no adjudin (see Table 2). At specified time points with = 3–5 rats for controls, and all treated rats in the three treatment groups, testes were collected and the body and testis weights were recorded. In each animal to be used for morphological and biochemical analysis, one of the testes was fixed in Bouin’s fixative (Polysciences, Inc., Warrington, PA, USA) to be used for subsequent histological analysis by haematoxylin and eosin staining using paraffin sections, the other testis was frozen in liquid nitrogen and stored at −80 °C to be used for lysate preparation for immunoblotting and to prepare frozen sections for dual-labelled immunofluorescence analysis. A description of rat usage for the three regimens and the control group for different experiments is listed in Table 2. It must be noted that in the animal groups at 6-, 20- and 30-week, while only one rat was dedicated for histological analysis and subjected to haematoxylin and eosin staining following paraffin embedding and sectioning as shown in Fig. 2, one of the testes from each animal used for BTB integrity assay (= 2 rats) was snap-frozen in liquid nitrogen and was also used to process for histological analysis, but using frozen sections and Mayer’s haematoxylin staining (and also for biochemical analysis) with the other testis used to assess the diffusion of fluorescence tag across the BTB, thus = 3 rats for these time points for morphological and biochemical analyses, and data from all three rats were consistent for each time point unless specified otherwise.

Table 2.   Animal usage for the three treatment regimens vs. control group
Time after treatmentControl groupAdjudin at 50 mg/kg b.w. (low-dose group)Adjudin at 125 mg/kg b.w. (high-dose group)Adjudin at 250 mg/kg b.w. (high-dose group)
Morphological and biochemical analysisaMorphological and biochemical analysisaBTB integrity assaybMorphological and biochemical analysisaBTB integrity assayMorphological and biochemical analysisaBTB integrity assay
  1. aFor the testes from rats specified for morphological and biochemical analysis, one of the testes from each rat was snap-frozen immediately in liquid nitrogen and stored at −80 °C until used for: (i) lysate preparation (to quantify the steady-level of spermatogonial markers and other pertinent BTB proteins) and (ii) preparation of frozen sections to score the number of spermatogonia/spermatogonial stem cells, and localization of BTB proteins by dual-labelled immunofluorescence analysis. The other testis of the same rat was fixed in Bouin’s fixative to be used to prepare paraffin sections for histological analysis following haematoxylin and eosin staining. For fertility tests, rats used for this test at specified time points reported in Fig. 1 were obtained from rats in the ‘Morphological and biochemical analysis’ group, and housed with the corresponding female rats (∼270 g b.w.) for 4 days before they were terminated for analysis. In some selected groups, such as rats from 20, 24 and 30 weeks, they were grouped and counted as a single group of 20/24-week for biochemical analysis, or 24/30-week for morphological analysis.

  2. bFor BTB integrity assay, = 2 rats for each treatment group, we also included a positive control group (= 2 rats) wherein rats were treated with CdCl2 at 3 mg/kg b.w. body weight (i.p.) which is known to induce BTB disruption. Also included is a negative untreated control group with = 2 rats in each BTB integrity assay. Thus, four additional rats (in brackets) were used for the two control groups in each BTB integrity assay.

  3. cFor morphological analysis, a testis from one rat was used for paraffin embedding and sectioning for H&E staining for histological analysis, and the other testis was snap-frozen in liquid nitrogen to be used for lysate preparation for immunoblot analysis. However, one testis was also removed from each of the two rats that were used for BTB integrity assay (with the other testis for the BTB integrity assay) and snap-frozen in liquid nitrogen, frozen sections were obtained, fixed in Bouin’s fixative, and stained with Mayer’s haematoxylin for histological analysis to assess the status of spermatogenesis; or for lysate preparation for immunoblot analysis. Thus, it is noted that n = 3 in these groups for morphological and biochemical analysis. This was done to minimize the number of rats used for each analysis and each experiment to avoid unnecessary killing of laboratory animals, yet sufficient number of replicates (and animals) could be obtained for meaningful statistical analysis. Nonetheless, a total of 214 adult rats were used for experiments in this report excluding ∼50 rats used for some preliminary and pilot experiments.

Time 0332 (4)32 (4)32 (4)
6 h 3 3 3 
9 h 3 3 3 
12 h 3 3 3 
1 days 3 3 3 
2 days 3 3 3 
4 days23 3 3 
7 days 3 3 3 
2 weeks232 (4)32 (4)32 (4)
6 weeks 1c2 (4)1c2 (4)1c2 (4)
8 weeks 2 2 2 
12 weeks33 3 3 
20 weeks 1c2 (4)1c2 (4)1c2 (4)
24 weeks 2 2 2 
30 weeks31c2 (4)1c2 (4)1c2 (4)
Total number of rats used13373037303730
image

Figure 2.  Histological analysis of testes in low-dose and high-dose adjudin-treated groups vs. controls to monitor changes in the status of spermatogenesis. Adult rats (∼300 g b.w.) were treated with a single low-dose or high-dose of adjudin at 50 or 250 mg/kg b.w., respectively, at time 0 (controls). Thereafter, rats (= 3) were terminated at specified time points for histological analysis using paraffin sections by haematoxylin and eosin staining. (A) Cross-sections of testes from the low-dose group with rats terminated at 0 (a), 6/8 (i.e. 6 or 8) (b), and 12 (c) hours (h); 1/2 (i.e. 1 or 2) (d), 4 (e), and 7 (f) day; and 2 (g), 6 (h), 12 (i), 20 (j), 24 (k), 30 (l) weeks. (B) Cross-sections of testes from the high-dose group. Germ-cell sloughing was detected in 6- to 8-h after adjudin administration at 50 or 250 mg/kg b.w. By 2 weeks, >98% of the tubules were devoid of germ cells in both treated groups. By 20 weeks, germ-cell repopulation was detected in most tubules in rats from the low-dose group and >75% of the tubules displayed normal spermatogenesis; however, tubules from rats in the high-dose group remained devoid of germ cells by 30 weeks. Scale bar = 100 μm, which applies to all micrographs in all panels. These micrographs are the results of a representative experiment, two additional experiments using frozen sections of testes from different sets of rats confirmed these observations.

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BTB integrity assay

The BTB integrity in vivo in rats was assessed by an assay established in our laboratory as earlier described (Li et al., 2006), which is based on the ability of an intact BTB to block the diffusion of a small fluorescence tag from the basal to the apical compartment of the seminiferous epithelium. Rats were under anaesthesia with ketamine HCl (60 mg/kg b.w.) together with an analgesic xylazine (10 mg/kg b.w.), which were administered intramuscularly (i.m.). A small incision, about 1 cm, was made in the area over the jugular vein to expose the blood vessel, and approximately 1.5 mg FITC-conjugated inulin (Mr 4.6 kDa) (Sigma-Aldrich) in 300 μl PBS was administered into the jugular vein with a 28-gauge needle. Animals were allowed to recover for about 30–45 min. Thereafter, rats were euthanized by CO2 asphyxiation. Testes were removed immediately and snap-frozen with liquid nitrogen. Testes were then embedded with Tissue-Tek OCT (optimal cutting temperature) compound (Sakura Finetek USA, Inc., Torrance, CA, USA) and frozen sections (10 μm) were obtained using a Microm HM500M microtome (ThermoScientific, Rockford, IL, USA) at −20 °C, placed on microscopic slides, air-dried and the distribution of inulin-FITC in the seminiferous epithelium was visualized using an Olympus BX61 Fluorescence Microscope (Olympus America Inc., Center Valley, PA, USA). Images were captured using an Olympus DP71 12.5 Mpx digital camera with Olympus MicroSuite Five software (version 1226) in an HP xw8400 Workstation running under Windows XP Pro and saved in TIFF format. Rats treated with CdCl2 at ∼3–5 mg/kg b.w. i.p. for 3 days were used for the BTB integrity assay as described above to serve as positive control while normal rat testes served as negative control. Treatment and control groups were processed in the same experimental session for comparison. The distribution of FITC (green fluorescence) in the seminiferous epithelium among tubules was monitored by fluorescence microscopy. BTB was considered damaged when fluorescence signal was no longer confined in the basal compartment, but found in the adluminal compartment. BTB integrity was semi-quantified by the distance travelled by the fluorescence signal from the basement membrane vs. the diameter of the seminiferous tubule. Each time point from each treatment group contained two rats and at least 90 tubules were randomly scored from each rat.

Immunoblot analysis

Lysates of testes were prepared in IP (immunoprecipitation) lysis buffer [50 mm Tris, pH 7.4, at 22 °C, containing 0.15 m NaCl, 1% Nonidet P-40 (vol/vol), 1 mm NaCl EGTA, 2 mm NaCl N-ethylmaleimide, 10% glycerol (vol/vol)] supplemented with protease inhibitor mixture (Sigma-Aldrich) and phosphatase inhibitor mixture I and II (Sigma-Aldrich) at a dilution of 1 : 100 to block protease and phosphatase activities as described (Lie et al., 2010a,b). Protein concentration was estimated using a BioRad Dc Protein Assay kit and a BioRad Model 680 Spectrophotometry Reader (BioRad Laboratories, Hercules, CA, USA). These lysates were used for immunoblot analysis with ∼100 μg protein for each sample. Proteins were transferred to nitrocellulose membranes and detected by immunoblotting with antibodies listed in Table 1. Chemiluminescence was performed using ECL kits (GE Healthcare, Waukesha, WI, USA) and a FujiFilm LAS-4000 Mini Luminescent Image Analyzer (FujiFilm Medical Systems USA, Inc., Stamford, CT, USA). Actin served as a protein loading control. All samples within an experimental group were processed simultaneously to avoid inter-experimental variations with = 3–4 independent experiments.

Dual-labelled immunofluorescence analysis

Frozen sections (7 μm) obtained in a cryostat at −20 °C were fixed in either Bouin’s fixative or 4% paraformaldehyde [wt/vol in PBS (10 mm sodium phosphate, 0.15 m NaCl, pH 7.4 at 22 °C)] for 10 min. Sections were then permeabilized with 0.1% Triton X-100 in PBS (vol/vol) for 4 min. Sections were blocked using 5% goat serum (vol/vol) with 0.1 BSA (wt/vol) in PBS, or 1% BSA (wt/vol) in PBS alone for 30 min, to be followed by an overnight incubation with primary antibodies diluted in corresponding blocking solution at 4 °C or room temperature. Sections were then incubated with Alexa Fluor-conjugated secondary antibodies [such as red fluorescence, Alexa Fluor 555; green fluorescence, Alexa Fluor 488 (Invitrogen, Carlsbad, CA, USA)] at 1 : 250 diluted with the corresponding blocking solution at room temperature for an hour. Sections were mounted with Vectorshield Antifade mounting media (Invitrogen) with DAPI (4′,6-diamidino-2-phenylindole, a fluorescent stain that bound to A-T rich regions in DNA) for fluorescence microscopy. Fluorescence images were captured using an Olympus DP71 12.5 Mpx (megapixel) digital camera interface to an Olympus BX61 fluorescence microscope using the Olympus MicroSuite Five Imaging software package (Version 1226) to obtain images in TIFF format. Image overlay and analysis were performed using PhotoShop in Adobe Creative Suite Design Premium software package (Version 3.0; Adobe Systems, San Jose, CA, USA). All staining experiments were performed 2–3 times with different sets of testes. The number of Plzf- and Utf1-positive cells per cross-section of seminiferous tubule was quantified as described below. To reduce inter-experimental variations, testes from all time points within a treatment group vs. controls were processed simultaneously in a single experimental session. Negative control included the use of mouse or rabbit IgG diluted in PBS to substitute the primary antibody to confirm that the immunofluorescence staining is not the result of an artifact.

Morphometric analysis

Morphometric analysis that scored Plzf and Utf1 positive cells in the seminiferous epithelium of tubules from rats from both control and the three treatment groups was performed essentially as earlier described (Wikström et al., 2004; Bascian et al., 2008; van Bragt et al., 2008; Pan et al., 2009; Forgione et al., 2010) with minor modifications. In brief, serial frozen sections (7 μm) of testes were used for scoring the number of Plzf and Utf1 positive cells by dual-labelled immunofluorescence analysis as follows. Each testis was first cut into two halves horizontally at the median line and each tissue block was mounted onto the cryostat. During serial sectioning, every fifth section was removed and collected onto a microscopic slide inside the cryostat at −20 °C, and a total of five sections were collected from each testis of a treated or control rat. Thus, the data presented herein for each time point are the representative result from a total of 15 cross-sections from three testes. Plzf and Utf1 positive cells were scored from randomly selected ∼60–80 seminiferous tubules in ∼20 fields using the 10× Objective in the Olympus BX 61 fluorescence microscope. The Plzf and Utf1 positive cells were further verified using the 40× Objective to confirm that the fluorescence staining is not the result of an artifact. We selected only cross-sectioned (i.e. round shaped) seminiferous tubules for scoring the Plzf and Utf1 positive cells instead of obliquely sectioned tubules for scoring in all treatment and control groups.

Fertility tests

Each male rat from the low-dose and high-dose groups vs. controls at specified time point was housed separately with a virgin female rat (∼300 g b.w.) for 4–5 days. The female rats were then housed separately for 21 days to allow the completion of gestation period (21 days) with free access to water and standard chow. The number of pups was recorded and any changes in gross morphology were noted.

General methods and statistical analysis

Each experiment was repeated at least three times or each data point had three to five animals. Statistical analysis of data for Plzf- and Utf1-positive cells scoring was performed with GB-STAT software package (Version 7.0; Dynamic Microsystems Inc., Silver Spring, MD, USA) using one-way anova followed by Dunnett’s test as described (Yan et al., 2008). Data derived from immunoblot analysis and BTB integrity assays in which the distance travelled by the fluorescence tag from the basement membrane to the apical compartment in a tubule versus the radius of the tubule (to provide semi-quantitative analysis) were analyzed statistically by two-way anova to be followed by Newman–Keul’s test.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Differential effects of low-dose and high-dose adjudin on the re-initiation of spermatogenesis

Previous studies have shown that adjudin (Fig. 1A) at 50 mg/kg b.w. (by gavage) induced transient infertility in rats by depleting germ cells from the seminiferous epithelium (Cheng et al., 2005; Mok et al., 2011). Thereafter, spermatogenesis resumed and germ cells gradually repopulated the entire epithelium of the seminiferous tubules, suggesting that SSCs were preserved after being challenged by adjudin. However, when adjudin was administered at multiple doses, fertility failed to rebound in a small percentage of rats (Cheng et al., 2005; Mok et al., 2011). These findings seemingly suggested that SSCs in some rats might have been depleted and/or other factors, which were crucial to spermatogenesis, were disrupted following treatment of rats with adjudin at multiple doses. Herein, the effects of a single high dose of adjudin at 125 or 250 mg/kg b.w. to spermatogenesis vs. 50 mg/kg b.w. on fertility were examined. After administration a single dose of 50, 125 or 250 mg/kg b.w. of adjudin to adult rats (∼300 g b.w. at time 0, by gavage) vs. controls (normal or vehicle only), a decrease in testis weight, but not body weight, was detected (Fig. 1B:a–b). It is noted that all the rats used in the present study had free access to standard rat chow and water ad libitum without dietary restriction. Changes in their body weight over time, such as reaching ∼700–750 g at the end of the study period by 30 weeks as reported herein is consistent with earlier findings (Hubert et al., 2000). It was also noted that by 12-week post-treatment, testis weight started to rebound gradually in the low-dose-treated group, but not in the high-dose-treated group (Fig. 1B:b), which is likely the result of the re-initiation of spermatogenesis in rats but only in the low-dose group. These findings are consistent with results of the fertility tests (Fig. 1C), because fertility rebounded in the low-dose group, but not in the two high-dose groups (Fig. 1C: a vs. b and c). It is noted that by 2-week post-treatment, rats remained fertile in the high-dose group (Fig. 1C:c) because of the sperm reserve in the epididymis, as adjudin had no effects on epididymal spermatozoa (Cheng et al., 2005).

image

Figure 1.  Changes in body weight, testis weight and fertility in rats treated with a low-dose and high-dose adjudin. (A) The structural formula of adjudin [1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide, C15H12Cl2N4O, Mr 335.18]. (B) Effects of adjudin on body weight (B:a) and testis weight (B:b) vs. control groups in which rats were treated with adjudin at low-dose (50 mg/kg b.w.) or high-dose (125 or 250 mg/kg b.w.) by gavage. Each bar = mean ± SD of = 3–6 rats. There was no significant change in body weight among the three groups, but there was a decrease in testis weight in both low-dose and high-dose groups beginning by 4-day post-treatment. Testis weight in the low-dose group, however, started to rebound by 6 weeks, but not in the high-dose group. (C) Results of fertility tests of control group vs. low-dose and high-dose-treated groups. The litter size is the number of pups that was counted with a male:female ratio of about 1 : 1, and no abnormal gross morphologies were detected in all pups. Fertility of rats in the low-dose-treated group rebounded by 30-week, but not in the two high-dose groups.

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The results reported in Fig. 1 that illustrate changes in testicular weight and the status of fertility in rats from the low-dose and high-dose groups vs. controls are consistent with findings of histological analysis shown in Fig. 2 which assessed the status of spermatogenesis. Depletion of germ cells following adjudin treatment in the low-dose (Fig. 2A) and high-dose (Fig. 2B) groups was detected as early as 6- to 8-h following treatment as immature germ cells were found in the tubule lumen, and by 2 weeks, the tubules were mostly devoid of germ cells. However, spermatogenesis resumed in the low-dose group beginning 20 weeks and by 30 weeks, >75% of the tubules displayed normal spermatogenesis (Fig. 2A:l vs. b-k and a) and fertility also rebounded in these rats (see Fig. 1C:a vs. b, c). However, resumption of spermatogenesis was not detected in the high-dose group by 30 weeks (Fig. 2B:l vs. b-k and a) and these findings are also consistent with the fertility status of this animal group (Fig. 1C: b, c vs. a).

Adjudin-induced germ-cell loss and infertility in both low-dose and high-dose adjudin-treated groups are associated with a surge in the expression of SSC/spermatogonial markers

The ability of spermatogenesis to recover in the low-dose-treated group suggests that spermatogonia, especially SSCs, were not depleted from the epithelium after adjudin treatment. On the other hand, failure of spermatogenesis to re-initiate in high-dose-treated group was likely due to a depletion of SSCs and spermatogonia from the epithelium. To assess any changes and/or loss in spermatogonia and SSCs, several putative markers for early A spermatogonia that included SSCs were used (Phillips et al., 2010) for immunoblot analysis. These include promyelocytic leukaemia zinc finger protein [Plzf, a transcriptional repressor essential for SSC self-renewal (Buaas et al., 2004)], undifferentiated embryonic transcription factor 1 [Utf1, a transcription factor involved in embryonic stem-cell differentiation (van den Boom et al., 2007)], octamer-4 [Oct4, a transcription factor essential for maintaining pluripotency and self-renewal properties of SSCs (Dann et al., 2008)] and thymus cell antigen-1 [Thy-1, a surface antigen of SSCs (Kubota et al., 2003)]. Immunoblotting results shown in Fig. 3A indicated that in both treatment groups, the steady-state levels of the four spermatogonial markers were significantly induced at the time of adjudin-induced germ-cell loss from the seminiferous epithelium (see Fig. 2 vs. Fig. 3). When virtually all the advanced germ cells (e.g. elongated/elongating spermatids, round spermatids, spermatocytes) were depleted from the seminiferous epithelium by 2- to 20/24-week after adjudin treatment (see Fig. 2), results shown in Fig. 3A and B (left panel) could be an over-estimate since there was an increase in the proteins being contributed by spermatogonia/SSCs and were analysed in these samples. Therefore, results shown in the left panel in Fig. 3B based on the scanned data of Fig. 3A were corrected for changes in the testis weight (see Fig. 1B: b) and expressed as protein content per pair testes and shown in the right panel in Fig. 3B. It is noted that the fold of increase in these four spermatogonial markers shown in Fig. 3A could not be accounted for by changes in the cellular composition being analysed by immunoblottings (Fig. 3B, right panels). Collectively, these findings suggested that spermatogonia/SSCs in the high-dose group are not depleted and may not be significantly different from the low-dose group. The increase in the spermatogonial markers’ expression might be attributed to a physiological response in the testis to maintain the germ line stem-cell population and at the same time, there is a need for them to differentiate for the re-initiation of spermatogenesis. Thus, markers pertinent to the maintenance of self-renewal (e.g. Plzf, Oct4) and markers related to differentiation (e.g. Utf1) were up-regulated in both groups, possibly to maintain the spermatogonia/SSC population.

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Figure 3.  Changes in the steady-state protein levels of SSC and spermatogonial markers in low-dose and high-dose-treated groups vs. normal rats at time 0. (A) Rats treated with adjudin at 50 or 250 mg/kg b.w. were terminated at specified time points to obtain lysates of testes. Immunoblots of SSC/spermatogonial markers Plzf, Utf1, Oct4 and Thy-1 using lysates from low-dose and high-dose-treated groups with actin serving as a loading control. This is a representative set of data from three experiments. (B) Histograms summarizing immunoblot analysis data of (A) are shown on the left panels which are plotted by normalizing each target protein (e.g. Plzf, Utf1) against actin. Protein levels at 0 h were arbitrarily set as 1. Right panels in (B) are histograms plotted by using data sets from the left panels, but corrected against the declining testis weight (see Fig. 1B) and expressed as relative protein content per pair testes. Each bar = mean ± SD (= 3). *< 0.05; **< 0.01.

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Both low-dose and high-dose adjudin-treated groups maintain similar number of SSC in the testes regardless of their ability to re-initiate spermatogenesis

The number of SSCs/spermatogonia in both low-dose (Fig. S1) and high-dose (Fig. 4A–F) groups vs. controls was estimated by dual-labelled immunofluorescence analysis using specific antibodies against Plzf and Utf1 (see Table 1) to score Plzf- and Utf1-positive cells in cross-sections of testes. It is noted that the numbers of Plzf- and Utf1-positive cells were ∼3 and 1 per cross-section of tubules in normal rat testes reported herein (Fig. 4G:i–ii) and this is consistent with an earlier report (van Bragt, et al. 2008). It is noted that the number of Plzf-positive cells representing As, Apr and long chain of Aal spermatogonia (up to 16 interconnected cells) (Suzuki et al., 2009) in both treatment groups increased transiently by ∼1.5-fold on day 2 post-treatment (Figure S1 and Fig. 4A–G). Thereafter, the number of Plzf-positive cells in the low-dose group declined gradually to ∼2 by week 20 and maintained at a similar level until week 24/30, and this number was not significantly lower than that of the control (∼2 vs. ∼3 Plzf-positive cells per cross-section to tubule between low-dose and control groups) (Fig. 4G:i); however, in high-dose group, the number of Plzf-positive cells was significantly reduced to ∼1.2 by week 24/30. (Fig. 4G:ii). On the other hand, in both low-dose and high-dose groups, the number of Utf1-positive cells which were a subpopulation of Plzf-positive cells (van Bragt et al. 2008) also showed a transient increase (∼3-fold) on day 2 post-adjudin treatment (Fig. 4G:i vs. ii). Thereafter, the number of Utf1-positive cells in both groups gradually decreased to ∼1, which was similar to that of control (Fig. 4G:i, ii). As Utf1-positive cells represent less advanced spermatogonia which are the As, Apr and short chain of Aal spermatogonia including the SSCs (Oatley & Brinster, 2006), it is likely that the number of SSCs was not reduced in both low- and high-dose treatment groups vs. control group. Thus, the re-initiation of spermatogenesis via spermatogonial differentiation to repopulate the seminiferous epithelium of the tubules detected in the low-dose group is not simply the result of the preservation of SSCs.

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Figure 4.  Changes in the population of SSC/spermatogonia in the testis by scoring Plzf- and Utf1-positive cells per cross-section of tubule in rats from the low-dose vs. high-dose adjudin-treated groups. (A–F) Representative images illustrating the number of Plzf-(green) and Utf1- (red) positive cells per cross-section of tubule from rats terminated at specified time points after administration of adjudin at 250 mg/kg b.w. Magnified views of rectangular boxed area in (a–d) are shown in (e–h). Square boxes in selected micrographs in (a–d) are magnified and placed in the same panel with corresponding Roman numerals. (G) Number of Plzf- and Utf1-positive cells per cross-section of tubule was counted using frozen sections of testes at specified time points from rats in the low-dose (G:i) vs. high-dose (G:ii) group. A transient increase in the number of Plzf- and Utf1-positive cells was observed in both groups 2 days after adjudin treatment. Thereafter, the number of Plzf-positive cells decreased gradually to ∼2 in G:i and G:ii ∼1.2 per tubule (vs. ∼3 in control). By 20-week, the number of Plzf-positive cells was not significantly different from control until 24–30 (24/30) weeks in the low-dose group (G:i), but remained mildly lower in the high-dose group (G:ii). By 4-day after treatment, the number Utf1-positive cells started to decline gradually in both groups and similar to that of control until 24–30 (24/30) weeks after treatment. Each bar = mean ± SD (= 2–3 rats per time point) and a total of 120–160 tubules were randomly scored from each rat. Scale bar = 100 μm in (A:a) applies to b–d; scale bar = 25 μm in (A:e) which applies to f–h. *< 0.05; **< 0.01.

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Effects of low-dose and high-dose adjudin treatment on the expression of constituent proteins at the BTB

Besides SSCs, earlier studies have demonstrated the significance of the BTB in spermatogenesis (Setchell & Waites, 1975), we thus examined the status of the BTB in both treatment groups vs. control rats. Immunoblot analysis was used to estimate the steady-state levels of various tight junction (TJ) (e.g. occludin, claudin-11) and basal ectoplasmic specialization (basal ES) (e.g. N-cadherin) proteins, which are integral membrane proteins at the BTB for its maintenance. We also examined changes in apical ES proteins (e.g. N-cadherin, β1-integrin and JAM-C) which are restricted to the Sertoli cell-elongating spermatid interface at the apical ES. It was shown that the steady-state levels of BTB proteins as well as those of the apical ES proteins were up-regulated in both adjudin treated groups vs. controls (Fig. 5A, Figure S2A,B), even when the changes in testicular weight were taken into consideration because of the changes in cellular composition in the samples being analysed (see Figure S2A vs. B, right panel). Although BTB proteins were up-regulated in both treatment groups, they failed to maintain the BTB integrity (see below).

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Figure 5.  Changes in the steady-state levels of TJ and apical ES proteins in the testes of rats from the low-dose vs. high-dose adjudin-treated group. Rats treated with either 50 (A) or 250 (B) mg/kg b.w. of adjudin were terminated at specified time points to obtain lysates of testes for immunoblot analysis of BTB proteins: occludin, claudin-11, N-cadherin, and apical ES proteins: N-cadherin, β1-integrin, JAM-C, with actin serving as a loading control. The results shown here are representative data from three independent experiments. Histograms summarizing results from (A) to (B) from three independent experiments from different rats in which changes in the steady-state level of a target protein was normalized against actin, together with statistical analysis are shown in Fig. S2 (see Supporting Information).

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Effects of low-dose and high-dose adjudin treatment on the localization of constituent proteins at the BTB

Dual-labelled immunofluorescence analysis was used to examine changes in the localization of occludin and ZO-1 in the seminiferous epithelium (note: occludin and ZO-1 is a major cell adhesion complex at the BTB with occludin serving as an integral membrane protein that binds to the adaptor protein ZO-1, which anchors the protein complex to the actin-based cytoskeleton). In normal rat testes, occludin (red fluorescence) and ZO-1 (green fluorescence) co-localized near the basement membrane consistent with their localization at the BTB (Fig. 6A,B). However, by 6–12 weeks in both low-dose (Fig. 6A) and high-dose (Fig. 6B) groups, occludin (as well as ZO-1, but to a lesser extent) was found to become mislocalized, diffusing away from the BTB site. However, the mislocalization of occludin and ZO-1 in the low-dose group was transient as by 20- and 24/30-week post-treatment, both BTB proteins redistributed to the original BTB site, making them indistinguishable from control rats (Fig. 6A). Yet, in the high-dose group, the mislocalization of both occludin and ZO-1 persisted until 20- to 24/30 weeks (Fig. 6B vs. A) and failed to recover. This trend was identical when another BTB adhesion protein complex claudin-11-ZO-1 was investigated in both the low-dose and high-dose groups vs. controls (see Figure S3). This pattern of changes in protein localization at the BTB seemingly suggest that there was a transient and irreversible disruption of BTB at the low-dose and high-dose group, respectively.

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Figure 6.  Changes in the cellular localization of occludin and ZO-1 in the seminiferous epithelium of rats from the low-dose and high-dose adjudin-treated groups vs. normal (control) rats. (A) and (B) illustrate the co-localization of occludin (red) and ZO-1 (green) in frozen sections of testes obtained from rats treated with adjudin at 50 (low-dose) or 250 (high-dose) mg/kg b.w., respectively. Occludin was found to co-localize with ZO-1 at the BTB in the seminiferous epithelium from tubules in control testes (A:a–d; B:a–d). In both treated groups, occludin and ZO-1 were found to redistribute from their original site and they appeared to become diffused and thickened within ∼1-day after treatment. This pattern of changes in redistribution for occludin and ZO-1 became more obvious by 6–12 weeks. However, in the low-dose group, the localization of occludin and ZO-1 was indistinguishable from controls by 20–24/30 weeks. But this shifted redistribution of occludin and ZO-1 persisted until 30 weeks in testes of rats from the high-dose group. The data shown herein are representative results of an experiment, which was repeated three times using samples from different sets of rat testes with each experiment yielded similar results. Bar in A:a (and B:a) = 50 μm, which applies to all other micrographs in A and B, respectively. This pattern of changes is also found for the claudin-11 and ZO-1 protein complex in the seminiferous epithelium in low-dose and high-dose adjudin-treated groups vs. normal (control) rats (see Fig. S3).

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A study by using an in vivo assay to assess the BTB integrity in the low-dose-treated group vs. high-dose-treated group

We hypothesized that the resumption of spermatogenesis in the low-dose group relied on a functional BTB, which rebounded following its transient disruption; however, the disrupted BTB in the high-dose group failed to recover, thereby impeding the re-initiation of spermatogenesis. To test this hypothesis, an in vivo BTB integrity assay was used to assess the BTB integrity in both treatment groups vs. controls. It was noted that by 2-weeks post-adjudin treatment, the BTB in rats from the low-dose-treated group remained intact (Fig. 7A), similar to the control, as no fluorescence signal was found in the apical compartment of the epithelium illustrating the BTB integrity (Fig. 7A: a, f vs. c, h). This finding is consistent with earlier results showing that the BTB of rats was not perturbed within 2 weeks after adjudin treatment at 50 mg/kg b.w. (Su et al., 2010). However, in the high-dose groups (rats administered adjudin at 125 or 250 mg/kg b.w.), BTB was found to be disrupted by 2 weeks after adjudin treatment. This was demonstrated by the presence of fluorescence signals in the apical compartment of the epithelium beyond the BTB, with some signals even found in the tubule lumen (Fig. 7A: d, i, e, j vs. a, f and b, g), similar to the positive controls in which rats were treated with CdCl2, which is known to disrupt the BTB irreversibly (Fig. 7A) (Setchell & Waites, 1970). By week 6, even in the low-dose-treated group, BTB was found to be disrupted (Fig. 7B). However, this disruption of BTB in the low-dose group was transient as BTB ‘re-sealed’ by week 20 and persisted to 24/30 weeks as shown by the ability of BTB at those time points to block the traverse of fluorescence signals from entering the apical compartment, indistinguishable from the controls (Fig. 7C and d vs. b and a). On the other hand, BTB integrity in the high-dose groups remained disrupted until weeks 24/30. These data are consistent with findings shown in Fig. 6 and Figure S3, suggesting that SSCs alone are not sufficient to re-initiate spermatogenesis.

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Figure 7.  A study to assess the BTB integrity in vivo following treatment of adult rats with adjudin vs. controls. Localization of inulin-FITC (green) in frozen sections of testes following administration of the fluorescence tag at the jugular vein to assess the BTB integrity that blocked its movement from the basal to the apical compartment in the epithelium. (A) Inulin-FITC was administered to rats at the jugular vein in normal rats at time 0 and the diffusion of inulin-FITC was monitored ∼45-min thereafter (a, negative control). Rats received CdCl2 (3 mg/kg b.w., via i.p.) at time 0 and used for BTB integrity assay on day 3 were served as positive control (b). Rat received adjudin at 50, 125 or 250 mg/kg b.w. were also used for the BTB integrity assay by 2-, 6-, 20- and 20–30 weeks after treatment and shown in (A) (c–e), (B), (C) and (D), respectively. In A, f–j are the magnified images of the corresponding images shown in a–e; and d–f are the corresponding magnified images in a–c of B–D. White broken-line circles in a–j from (A) and a–f from (B-D) indicate the relative location of the basement membrane in the seminiferous tubule, at the site of the BTB. White brackets (f–j in A; d–f in B–C) indicated the relative distance traveled by inulin-FITC from the BTB. (E) This histogram provides the semi-quantitative data of the BTB integrity assay by quantifying the distance traveled by inulin-FITC from the basement membrane at the BTB site vs. the radius of a tubule. Each bar = mean ± SD of 90 tubules that were randomly selected and scored from testes of two rats for each time point. Scale bar = 100 μm in a which applies to b–e in (A) and also applies to b–c in (B–D); scale bar = 50 μm in f which applies to g–j in (A); scale bar = 50 μm in d which applies to e–f in (B–D). *< 0.05; **< 0.01.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Toxicants (e.g. adjudin) that disrupt spermatogenesis and cause infertility do not impede the population of SSC/spermatogonia, but impair the functional status of the BTB

As reported herein, the testes from rats in both the low-dose and high-dose adjudin-treated groups displayed a phenotype in which virtually all the seminiferous tubules were devoid of germ cells, yet the number of Utf1-positive cell population, representing As, Apr and short chain of Aal spermatogonia (van Bragt et al. 2008), was maintained in both treatment groups at a level similar to that of normal adult rats, and was not reduced. However, the number of Plzf-positive cells in rats from the high-dose group was significantly lower than that of normal rats (reducing from ∼2.8 Plzf-positive cells/cross-section of tubule in normal rats to ∼1.2 Plzf-positive cells/cross-section of tubule in the high-dose group) by the end of the experimental period at 24/30-week post-adjudin treatment. Therefore, we speculate that the type of spermatogonia which had their number reduced was more advanced Aal spermatogonia. This speculation is reached since Plzf is expressed from As to long chain of Aal spermatogonia (Suzuki et al., 2009) which include more advanced spermatogonia, whereas the Utf1-positive cells which represent less differentiated spermatogonia (van Bragt et al. 2008) was not reduced in both treatment groups versus controls. Thus, the reduced population of Plzf-positive cells in the tubules from high-dose-treated group vs. control and the low-dose groups could be the result of spermatogonia (note: spermatogonia were not depleted in rats from the high-dose group as the population of Utf1-positive cells in the tubules was maintained) that failed to proceed beyond short chain of Aal. This thus hinders re-initiation of spermatogenesis, and we speculate that this is due to the unfavourable microenvironment in the basal compartment, perhaps at the stem-cell niche, for spermatogenesis because of the disruption of the BTB. For instance, ‘unwanted’ but yet-to-be identified signals originated in the apical compartment could no longer be ‘prevented’ from reaching the stem cell niche because the BTB had been disrupted. This argument was supported by the observations that the number of Plzf-positive cells in low-dose-treated group was also significantly reduced by 6/8-week and 12-weeks post-adjudin treatment, but their number rebounded when BTB was ‘resealed’ by 20 weeks and thereafter. Besides, this postulate is supported by earlier studies reporting that after exposure of rodents to various toxicants or irradiation to induce infertility, type A spermatogonia were still present in tubules of animals with azoospermia. These include rats treated with 2, 5-hexanedione (Boekelheide & Hall, 1991), irradiation (Kangasniemi et al., 1996), dibromochloropropane (Meistrich et al., 2003) and procarbazine (Meistrich et al., 1999). In the above studies, it was shown that although the number of spermatogonia was reduced, some of them survived the toxicant exposure and maintained a constant number as manifested by active proliferation of spermatogonia based on mitotic index and proliferation assay (Allrad & Boekelheide, 1996; Shuttlesworth et al., 2000). However, the proliferating spermatogonia rapidly underwent apoptosis and thus failed to differentiate beyond type A spermatogonia (Allrad & Boekelheide, 1996; Shuttlesworth et al., 2000). Unfortunately, the integrity of the BTB was not investigated in any of these earlier studies. Collectively, the findings reported herein and those reported earlier thus support the notion that the SSCs/spermatogonia in the testes following toxicant treatment [note: adjudin is a ‘toxicant’ in the sense that it affects germ-cell adhesion and spermatogenesis even though it did not cause any mortalities at up to 40 times of the effective dose (50 mg/kg b.w., by gavage) to induce infertility (Mruk et al., 2006)] can proliferate and differentiate to re-initiate spermatogenesis in an optimal microenvironment in the epithelium with an intact BTB.

A functional and intact BTB is crucial to re-initiate and maintain spermatogenesis

To the best of our knowledge, this is the first report demonstrating the physiological significance of BTB in re-initiating spermatogenesis after its challenge by toxicants. However, the importance of BTB in spermatogenesis is well established because it provides a specialized microenvironment in the apical compartment for post-meiotic germ-cell development by restricting the types and amounts of substances (e.g. ions, nutrients, hormones, electrolytes, biomolecules, water and others) that can have access to developing spermatids via paracellular transport. Besides, BTB also maintains cell polarity and confers immune privilege to the testis (Cheng & Mruk, 2010; Meinhardt & Hedger, 2010). In humans, the BTB has been implicated to affect fertility status (Landon & Pryor, 1981; Koksal et al., 2007). The importance of BTB in fertility is best demonstrated in mouse models with BTB integral membranes proteins (e.g. claudin-11 and occludin) being knocked out. For instance, claudin-11−/− mice were shown to have a disorganized Sertoli cell BTB and were infertile (Gow et al., 1999; Mazaud-Guittot et al., 2010), and spermatogenesis was later shown to fail to proceed beyond meiosis (Mazaud-Guittot et al., 2010). On the other hand, although occludin−/− mice were fertile in young adult males by 6 weeks of age (Saitou et al., 2000), and the fertility in these occludin−/− mice was maintained up to 10-week post-partum (Saitou et al., 2000; Takehashi et al., 2007); however, the seminiferous tubules in these occludin−/− mice were devoid of all spermatids and spermatocytes by age 36- to 60-week post-partum and these mice were infertile (Saitou et al., 2000; Takehashi et al., 2007) illustrating meiotic arrest as the result of a loss of occludin-based TJ-fibrils at the BTB. This delay in response to the knockout of occludin in these mice could be explained as follows. It is likely that the initial loss of occludin was superseded by other tight junction proteins (e.g. claudin-5, claudin-11, JAM-A, JAM-B) in young adult mice, but as animals aged, a permanent loss of occludin-based tight junction-fibrils could no longer be superseded, leading to an inability of the spermatogonia to differentiate beyond Aal.

In this study, it was shown that the BTB integral proteins claudin-11, occludin and N-cadherin were up-regulated during adjudin-induced germ-cell loss that led to infertility, yet they failed to strengthen the BTB. It is possible that the up-regulated occludin and claudin-11 proteins were mislocalized as revealed by the unusual thickening and diffusion of the fluorescence from the BTB site based on dual-labelled immunofluorescence analysis. Mislocalization of BTB proteins can be a sign of spermatogenic and BTB malfunction as demonstrated by studies showing that claudin-11 expression was also induced, but mislocalized in testes from infertile men with Sertoli cell only syndrome (Nah et al., 2010), as well as in testes of testicular intraepithelial neoplasia patients with loss of BTB function (Fink et al., 2009). Besides, there was also a study reporting that patients with carcinoma in situ having a perturbed BTB, which was manifested by a mis-localization of ZO-1, diffusing from the BTB site in the seminiferous epithelium (Fink et al., 2006), analogous to our findings in this report. Furthermore, an in vivo BTB integrity assay has demonstrated that the BTB from rats in the high-dose-treated group was disrupted at least beginning by 2-week post-adjudin treatment, and it was not recovered at the end of this study by 24/30 weeks. On the other hand, although the BTB in low-dose-treated group was once perturbed by 6-week post-treatment, it was ‘resealed’ by week 20 and this integrity was maintained until the end of this study by 24/30 weeks. In summary, while there is no reduction in the population of SSC/spermatogonia in both treatment groups, spermatogenesis failed to resume in the high-dose group because of the disrupted BTB, suggesting that a functional BTB is required to sustain spermatogonial differentiation to re-initiate spermatogenesis.

BTB integrity and infertility in men

In this context, it is of interest to note that following radiotherapy, chemotherapy using drugs such as procarbazine, or exposure to environmental toxicants like dibromochloropropane, prolonged azoospermia is detected in these men (Slutsky et al., 1999; Howell & Shalet, 2005). In these disease models, it is likely that the loss of fertility is the result of BTB disruption as spermatogonia are present in these patients. This is analogous to the cadmium model in which exposure of CdCl2 to rats leads to irreversible disruption of the BTB, which in turn, perturbs re-initiation of spermatogenesis (Wong et al., 2004). Also, in both rodents and humans, progression of meiosis and spermiogenesis occurs only after the BTB is established by age 15–17 days postpartum in rodents and following puberty in humans by age ∼11–14 years (Cheng & Mruk, 2010), illustrating the crucial significance of the BTB to initiation of spermatogenesis. Nonetheless, much research is needed to understand the underlying mechanism(s) by which a functional BTB regulates the initiation of spermatogonial differentiation and spermatogenesis. For instance, is it possible that the BTB ‘seals’ off the regulatory biomolecule(s) or factor(s) in the apical compartment that blocks spermatogonial differentiation (or the BTB regulates how much inhibitory substance(s) can reach the basal compartment to block spermatogonial differentiation)? This is physiologically necessary since there is a fixed number of Sertoli cells in the seminiferous tubules to provide structural and nutritional supports to developing germ cells, thus the events of meiosis/spermiogenesis and spermatogonial differentiation must be tightly regulated to avoid overwhelming the Sertoli cell capacity. A disruption of this barrier thus abolishes the necessary cross-talk between the apical and basal compartments, leading to a shut-down of spermatogonial differentiation.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

This work was supported by grants from the National Institutes of Health (NICHD, R01 HD056034, R01 HD056034-02S1 and U54 HD029990 Project 5 to CYC; R03 HD061401 to DDM), and Hong Kong Research Grants Council and CRCG of the University of Hong Kong (to WML). K.W.M. and C.Y.C. performed the research; C.Y.C. designed the research study; W.M.L. contributed essential reagents; K.W.M., D.D.M. and C.Y.C. analysed and critically evaluated the data; and K.W.M. and C.Y.C. wrote the paper.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Figure S1. Change in the population of SSCs/spermatogonia in the testis by scoring Plzf- and Utf1-positive cells per cross-section of tubule in rats from the low-dose adjudin treated group. (A-F) Representative images illustrating the number of Plzf- (green) and Utf1- (red) positive cells per cross-section of tubule from rats terminated at specified time points after administration of adjudin at 50 mg/kg b.w. Magnified views of rectangular boxed area in (a-d) are shown in (e-h). Square boxes in (a-d) are magnified and placed in the same panel with corresponding Roman numerals. The number of Plzf- and Utf1-positive cells per cross-section of tubule was counted using frozen sections of testes at specified time points from rats in the low-dose group and shown in Fig. 4G in the “main text”.

Figure S2. Change in the steady-state levels of TJ and apical ES proteins in the testes of rats from the low- versus high-dose adjudin treated group. Rats treated with either 50 (A) or 250 (B) mg/kg b.w. of adjudin were terminated at specified time points to obtain lysates of testes for immunoblot analysis of BTB proteins: occludin, claudin-11, N-cadherin, and apical ES proteins: N-cadherin, 1-integrin, JAM-C, from rats in the low- versus high-dose group with actin serving as a loading control. The results shown here are representative data from three independent experiments such as those shown in Fig. 5 (see main text). Histograms summarizing results from Fig. 5A and 5B are shown on the left panels in (A) and (B), respectively, which are plotted by normalizing each data point against actin. Protein levels at 0 hour were arbitrarily set as 1. Right panels are histograms plotted by each data point on the left panels correcting against the decreasing testis weight (see Fig. 1B) and expressed as relative protein level per pair testes. Bars = mean ± SD (n = 3 rats). *P < 0.05; **P < 0.01.

Figure S3. Changes in the cellular localization of claudin-11 and ZO-1 in the seminiferous epithelium of rats from the low- and high-dose adjudin treated groups versus normal (control) rats. (A) and (B) illustrate the co-localization of claudin-11 (red) and ZO-1 (green) in frozen sections of testes from rats treated with adjudin at 50- (low-dose) or 250- (high-dose) mg/kg b.w., respectively, with rats terminated at specified time points. Claudin-11 was found to co-localize with ZO-1 at the BTB in the seminiferous epithelium from tubules in control testes (A:a-d). In both treated groups, the localization of claudin-11 and ZO-1 was altered within ~1-day after adjudin treatment in both groups that became diffused from the BTB site and “thickened”. This “thickening” of claudin-11 and ZO-1 became more obvious by 6- to 12-week. However, in the low-dose group, the localization of claudin-11 and ZO-1 and their co-localization 2 rebounded by 20- to 30-week, making them indistinguishable from the control rat testes, but not in the high-dose group wherein claudin-11 and ZO-1 remained mist-localized. This trend of changes in localization for claudin-11 and ZO-1 is similar to occludin and ZO-1 shown in Fig. 8. The data shown herein are representative results of an experiment which was repeated three times using samples from different sets of rat testes and yielded similar results. Bar in A:a (and B:a) = 50 m, which applies to all other micrographs in the panel.

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