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From the * Department of Pathology & Cell
Biology, University of South Florida College of Medicine, Tampa, Florida; and
the Center for Biomedical Research, Population
Council, New York, New York.
| Correspondence to: Katja M. Wolski, Department of Pathology & Cell Biology, University of South Florida College of Medicine, 12901 Bruce B. Downs Blvd., MDC6, Tampa, FL 33612-4799 (e-mail: kwolski{at}hsc.usf.edu). |
| Received for publication April 25, 2006; accepted for publication June 21, 2006. |
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Abstract |
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Key words: Ectoplasmic specialization, testis, micropipette, adherens junction, male contraception
The ES is an apical cytoskeletal structure of the Sertoli cell associated with Sertoli-spermatid binding at the adherens junction (AJ) (Russell, 1977b; Russell, 1980). This structure is important in cell-cell adhesion in the seminiferous epithelium, ensuring the retention of spermatids as they mature into spermatozoa. It is believed that the ES forms to strongly anchor the step-8 spermatid to the seminiferous epithelium. In the rat, ESs are first seen at Stage VIII of spermatogenesis, the time when the step-8 spermatid appears, and are present at the AJ until appropriate release of the step-19 spermatid. Inappropriate release of spermatids (ie, spermatid sloughing) is related to abnormal ES structure and oligospermia (O'Donnell et al, 1996; O'Donnell et al, 2000). A reduction of mature sperm in semen (Russell et al, 1988; Boekelheide et al, 1989; O'Donnell et al, 1996; O'Donnell et al, 2000) and conditions associated with oligospermia are associated with structurally abnormal or absent Sertoli ESs (Cameron and Griffin, 1998).
A number of health-related conditions are associated with reduced fertility potential and oligospermia in men, including varicocele (Cameron and Snydle, 1980), hyperprolactinemia (Cameron et al, 1984), diabetes (Murray et al, 1983), and idiopathic oligospermia (Cameron and Griffin, 1998). These conditions are all associated with reduced sperm in the semen, ie, oligospermia, and ultrastructural pathology unique to the junctional apparatus of the seminiferous epithelium (Cameron and Griffin, 1998). Cap stage spermatids in the human (step-8 spermatids in the rat) are presumed to be tightly anchored to the seminiferous epithelium at a Sertoli cell AJ, which includes the unique Sertoli ES (Russell, 1977b; Russell, 1980). In both in vitro and in vivo observations of experimental animal models, disruption of this junction results in spermatid sloughing and subsequent oligospermia (Russell et al, 1988; Boekelheide et al, 1989; O'Donnell et al, 1996; O'Donnell et al, 2000).
Adjudin, formerly known as AF-2364 (1-(2,4-dichlor-obenzyl)-1H-indazole-3-carbohydrazide), depletes seminiferous tubules of germ cells (Cheng et al, 2001; Grima et al, 2001). By day 14 of administration of Adjudin, adult seminiferous tubules are found nearly devoid of elongated and round spermatids and spermatocyte numbers have been reduced, with no significant effect on reproductive hormone levels (Cheng et al, 2001). Although there has been speculation, it is not yet been determined whether adjudin works at the level of the adherens junction or the ES.
This project was designed to measure the strength of junctions between step-8 spermatids and Sertoli cells in the presence of various concentrations of Adjudin. To do this, a micropipette pressure transducing system was used to measure the force needed to detach step-8 spermatids from Sertoli cells (Wolski et al, 2005) in the presence of Adjudin (0 ng/mL, 1 ng/mL, 50 ng/mL, 125 ng/mL, or 500 ng/mL in EtOH) (Siu et al, 2003) and reproductive hormones (follicle-stimulating hormone and testosterone). It is hypothesized that Adjudin at higher concentrations will disrupt the STJC and cause reduced binding strength between the Sertoli cell and step-8 spermatid.
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Materials and Methods |
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Sertoli Cell Isolation, Culture, and Pretreatment
Briefly, testes were excised from prepubertal male rats, and the parenchyma
was digested using routine sequential enzymatic treatments with trypsin
(0.25%; Sigma Chemical Co, St Louis, MO) and collagenase (0.20%; BD, Franklin
Lakes, NJ). Isolated cells were plated to near confluence on 13-mm round glass
coverslips coated with 1:3 Matrigel (BD) in culture medium in 24-well cell
culture dishes. Cultures were incubated in DMEM:F12 medium [supplemented with
0.01 mol/L retinol (Sigma), 1000 µl/100 mL ITS (BD), 500 µL/500 mL
gentamicin (Sigma), and 5 mL/500 mL antibiotic/antimycotic (Cellgro;
Mediatech, Inc, Herndon, VA)] at 39°C in a humidified incubator with 5%
CO2-95% air for 48 hours to expedite the removal of contaminating
germ cells. After the 48-hour preincubation, the cultures were exposed to a
20-mM Tris-HCl buffer for 2.5 minutes to hypotonically lyse any remaining germ
cells, then incubated in supplemented DMEM:F12 at 33°C in a humidified
incubator with 5% CO2-95% air for 24 hours. After the 24-hour
incubation, the medium was replaced with supplemented DMEM:F12 containing 0.06
µg/mL follicle-stimulating hormone (FSH; NIDDK-oFSH-20, AFP7028D, 175
x NIH-FSH-S1) and 100 nM testosterone (T; Sigma) to optimize in vitro
Sertoli-spermatid binding. These pretreated Sertoli cell cultures were used in
the coculture experiments.
Round Spermatid Isolation and Unit Gravity Velocity Sedimentation
Prestep 9 spermatids (round spermatids) were isolated from an adult male
rat testis. Briefly, the decapsulated adult testis was digested with 0.10%
collagenase (37°C, 80 oscillations/min, 30 minutes; Gibco, Invitrogen,
Carlsbad, Calif) to separate seminiferous tubules from the testicular
interstitial tissue. The washed seminiferous tubules were then digested with
0.25% trypsin (37°C, 90 oscillations/min, 15 minutes; Sigma) to separate
the peritubular cells from the seminiferous epithelium and to expedite the
release of germ cells from the seminiferous epithelium. A 0.20% trypsin
inhibitor solution (Sigma) was added to terminate the trypsin reaction. The
resulting cell suspension (mixed germ cells and Sertoli cells) was resuspended
in 25 mL McCoy media + 0.5% BSA.
Using sterile technique, the gradient chambers on a STA-PUT velocity sedimentation cell separator were filled with the appropriate McCoy + BSA medium (2% and 4% BSA), and a linear gradient (2%–4%) was built under the cell suspension, at the loading rate initially at 10 mL/min. After 20 minutes, the rate was increased to 40 mL/min. Eighty minutes prior to the end of the collection time (4 hours), media with germ cell fractions were collected using a Fractomat automatic fraction collector (10 mL/vial at 160 drops/min). Round spermatids (prestep 9) were identified by phase contrast microscopy and pooled, washed, and resuspended in McCoy media. The number of cells in the spermatocyte and spermatid fractions were counted by hemocytometric analysis and assayed for viability by trypan blue exclusion.
Sertoli-Germ Cell Coculture
Approximately 400 000 isolated germ cells (round spermatid-enriched) were
added directly to the pretreated Sertoli cell-enriched monocultures. The
Sertoli-germ cell cocultures were incubated in a humidified chamber at
33°C with 5% CO2-95% air for 36 hours with 0.06 µg/mL FSH +
100 nM T to optimize Sertoli-spermatid binding, as previously described
(Cameron and Muffly, 1991;
Cameron et al, 1993).
Addition of Adjudin to the Coculture
After 30 hours of incubation, Vehicle 1 (2.5 µL EtOH) was added to 1
column of the 24-well plate and incubated for 1 hour at 33°C. After the
1-hour incubation time, the next column received 1 ng/mL Adjudin in 2.5 µL
EtOH and incubated for 1 hour at 33°C. This continued with the remaining
concentrations of Adjudin (50 ng/mL, 125 ng/mL, and 500 ng/mL)
(Siu et al, 2003) and ended
with Vehicle 2 (same as Vehicle 1) to ensure that time was not the factor
affecting the junction.
Measurement of Junctional Strength Using a Micropipette Pressure Transducing System (MPTS)
The Sertoli-germ cell cocultures were imaged on an inverted interference
contrast microscope (Axiovert 100, Zeiss) with a 20x objective. The
microscope was fitted with the MPTS, as previously described
(Wolski et al, 2005). After
the 1-hour incubation with the treatment, the cover slips were washed 5x
by gentle pipetting with supplemented DMEM:F12 + FSH and T (without Adjudin or
EtOH). Cover slips containing the Sertoli-germ cell cocultures were carefully
removed from the well. A step-8 spermatid were identified as a 10-µm round
cell containing an eccentric nucleus, as previously described
(Leblond and Clermont, 1952;
Cameron and Muffly, 1991). The
detachment of individual step-8 spermatids from Sertoli cells and subsequent
force measurement and analysis was performed as previously described
(Wolski et al, 2005). Briefly,
pressure at the tip of a 10-µm-diameter micropipette was controlled by a
system consisting of 2 water reservoirs and a pressure transducer connected
between the 2 reservoirs. To detach spermatids from Sertoli cells, the glass
micropipette tip was brought into close proximity to the unbound cell surface
of the spermatid, and the hydrostatic pressure required to detach it from the
underlying Sertoli cell monolayer was recorded on the transducer. The recorded
pressure (in cm-H2O) was used to calculate force via the equation F
= 
P · 
R2p, where F (pN) is the
force on a static cell, P is the suction pressure
(N/µm2), and R2p is the cross sectional
area of the pipette (µm2). To convert the pressure reading
received in cm-H2O to N/µm2 for use in the above
equation, the conversion factors 1 cm-H2O = 98.06 Pa and 1 Pa =
N/m2 were used, since the international unit of force is newtons (1
N = 1 kg m/s2), and the international unit of pressure is pascal
(Pa). Each detachment event (a maximum of 4) consisted of a 5-second suction
pressure interval. If the germ cell did not dissociate, the detachment effort
was abandoned, and the last pressure reading was recorded.
Viability/Cytotoxicity Assays
Sertoli cell cultures were prepared from 20-day-old rat testes by
sequential enzymatic treatments as described
(Cameron et al, 1987), and
cells were plated at high-density (0.5 x 106
cells/cm2) on Matrigel (diluted 1:7 with Ham's F-12 Nutrient
Mixture and Dulbecco Modified Eagle's Medium [F-12/DMEM], 1:1; Sigma)-coated
Nunclon 24-well dishes in F-12/DMEM supplemented with 10 µg/ml bovine
insulin, 5 µg/ml human transferrin, 10 µg/ml bacitracin, 2.5 ng/mL EGF,
0.06 µg/ml FSH, and 100 nM T. To obtain Sertoli cells with a purity greater
than 98%, cultures were hypotonically treated. Media were replaced every 24
hours thereafter, and Sertoli cells were incubated for an additional 3 days.
This was followed by the isolation of germ cells from 90-day-old rat testes,
as previously described (Aravindan et al,
1996; Aravindan et al,
1997). In this experiment, germ cell preparations were exposed to
successive glass wool filtration steps, and thus consisted of spermatogonia,
spermatocytes, and round and elongating spermatids when examined
microscopically. Germ cells were added directly to Sertoli cell cultures at a
Sertoli:germ cell ratio of 1:3 and cocultured for 36 hours
(Mruk et al, 1997).
Thereafter, cocultures were rinsed twice with media to remove unbound germ
cells and increasing concentrations of Adjudin (1, 50, 125, and 500 ng/mL and
1 µg/ml) were added. Sertoli-germ cell cocultures were incubated for 0, 1,
3, 6, and 12 hours. These cocultures were then used for viability/cytotoxicity
assays. Because viable cells are characterized by the presence of
intracellular esterase activity, this assay measured the ability of cells to
enzymatically convert nonfluorescent, cell-permeable calcein AM to fluorescent
calcein. Briefly, media was removed from Sertoligerm cell cocultures and cells
gently rinsed with media. Calcein AM (
2–5 µM, prepared in media
or PBS, pH 7.4 prior to immediate use to prevent hydrolysis; Invitrogen) was
added to Sertoli-germ cell cocultures and incubated briefly at 37°C or
room temperature. Fluorescence was quantified at 10–15-minute intervals
for up to 60 minutes at 485 nmEX and 535 nmEM using a
Tecan GENios fluorescence plate reader. Controls consisted of Sertoli-germ
cell cocultures cultured in the absence of Adjudin and in the presence of
vehicle (ethanol:DMSO, 1:1 dilution). Nonviable Sertoli-germ cell cocultures,
which lacked the ability to enzymatically convert calcein AM to calcein, were
prepared by treating cells with 75% ethanol (30 minutes) or 0.5% saponin (10
minutes).
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Results |
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Viability/cytotoxicity assays demonstrated that the viability of Sertoli-germ cell cocultures was not affected when these cells were incubated with increasing concentrations of Adjudin for up to 12 hours (Figure 2). Time points beyond 12 hours were not examined because higher doses of Adjudin (500 ng/mL and 1 µg/ml) perturb Sertoli-germ cell adhesion, resulting in a decrease in cell number in these wells.
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Discussion |
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100 the testes of treated animals were
indistinguishable from controls (Grima et
al, 2001). However, it was not known whether this compound
actually affects the strength of the STJC.
We have, for the first time, determined that the strength necessary to detach step-8 spermatids from a Sertoli cell monolayer is reduced by specific doses of Adjudin in vitro, indicating a functional alteration of the STJC. The data presented confirm the hypothesis that this compound, at higher concentrations, disrupts the STJC, causing weaker binding between the Sertoli cell and step-8 spermatid. Only the STJC between step-8 spermatids and Sertoli cells, which contains the ES, was tested. It is not known whether Adjudin affects the strength of desmosome-like junctions that are present, for example, between Sertoli and pachytene spermatocytes. Recent studies have suggested that Adjudin affects the ES by activating RhoB within hours of administration. This in turn activated ROCK, LIMK1, and cofilin, which perturbed actin cytoskeleton dynamics and resulted in germ cell detachment (Lui et al, 2003). Moreover, ß1 integrin, which is predominantly located at the apical ES, was also shown to be up-regulated following Adjudin treatment, further activating the FAK/PI 3-kinase/p130Cas/MAP kinase (Siu et al, 2003) pathway. Though the exact protein complex acting as the receptor for Adjudin has not yet been defined, it is thought that through these signaling pathways, changes in the polymerization and depolymerization of actin at the ES lead to a depletion of germ cells from the seminiferous epithelium, in particular round and elongating spermatids (Cheng et al, 2005). It should be noted that Adjudin is a chemical entity that shares structural similarities with lonidamine [1-(2,4)-dichlorobenzyl-1H-indazole-carboxylic acid], which is known to severely damage stress fibers (eg, actin filaments) in Sertoli cells (De Martino et al, 1981; Silvestrini et al, 1984). Likewise, preliminary studies have shown that Adjudin can induce extensive remodeling of the actin cytoskeleton in these cells (Mruk and Cheng, unpublished observations). What remains to be determined, however, is why Sertoli cell actin at the apical ES is sensitive to Adjudin's effects when this protein is a constituent of virtually all cell types. Certainly, other upstream regulators of RhoB activity, as well as additional signaling cascades, are likely to be involved, and their identification will help in determining why the apical ES is a primary target for Adjudin-mediated restructuring in the testis.
Results from this study show that the junctional strength between Sertoli cells and step-8 spermatids is reduced by Adjudin in vitro, supporting the potential use of this chemical as a male contraceptive.
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