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Biology of Reproduction 65, 1340-1351 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

A 22-Amino Acid Synthetic Peptide Corresponding to the Second Extracellular Loop of Rat Occludin Perturbs the Blood-Testis Barrier and Disrupts Spermatogenesis Reversibly In Vivo1

Nancy P.Y. Chunga, Dolores Mruka, Meng-yun Moa, Will M. Leeb, and C. Yan Cheng2,,a

a The Population Council, Center for Biomedical Research, New York, New York 10021 b Department of Zoology, University of Hong Kong, Hong Kong, China

ABSTRACT

When Sertoli cells were cultured in vitro on Matrigel-coated bicameral units, the assembly of the inter-Sertoli tight junction (TJ) permeability barrier correlated with an induction of occludin expression. Inclusion of a 22-amino acid peptide, NH2-GSQIYTICSQFYTPGGTGLYVD-COOH, corresponding to residues 209–230 in the second extracellular loop of rat occludin, at 0.2–4 µM into Sertoli cell cultures could perturb the assembly of Sertoli TJs dose-dependently and reversibly. This peptide apparently exerts its effects by interfering with the homotypic interactions of two occludin molecules between adjacent Sertoli cells at the sites of TJs, thereby disrupting TJs, which, in turn, causes a decline in transepithelial electrical resistance across the Sertoli cell epithelium. When similar experiments were performed using a 22-amino acid myotubularin peptide, NH2-TKVNERYELCDTYPALLAVPAN-COOH (residues 156–177), no effects on the assembly of inter-Sertoli TJs in vitro were noted. When a single dose of this synthetic occludin peptide was administered to adult rats intratesticularly at 1.5–10 mg/testis, germ cells began to deplete from the seminiferous epithelium within 8–16 days. By 27 days, virtually all tubules were devoid of germ cells. This antispermatogenic effect was reversible, because germ cells progressively repopulated the epithelium thereafter. Treated testes were indistinguishable from normal or control testes by 68 days post-occludin peptide treatment when assessed using histological analysis. In contrast, control rats receiving either no treatment, vehicle alone, or a 22-amino acid synthetic peptide of myotubularin displayed no changes in the testicular morphology at all time points. The occludin peptide-induced germ cell depletion was also accompanied by a disruption of the blood-testis barrier (BTB) when assessed by micropuncture techniques quantifying [125I]-BSA in rete testis fluid and seminiferous tubular fluid following i.v. administration of [125I]-BSA through the jugular vein. These results illustrate that the occludin peptide-induced disruption of the BTB may possibly affect the underlying adherens junctions, which causes premature release of germ cells from the epithelium and reversible infertility.

Sertoli cells, sperm, sperm maturation, spermatogenesis

INTRODUCTION

Throughout spermatogenesis, multiple molecular, biochemical, and cellular events take place concurrently in the seminiferous epithelium, leading to the formation of eight spermatids from a single type B spermatogonium (for reviews, see [1, 2]). In addition, inter-Sertoli tight junctions (TJs) that constitute the blood-testis barrier (BTB) must be disrupted and reassembled to allow the timely passage of pre- and leptotene spermatocytes from the basal compartment of the seminiferous epithelium, across the BTB, and entering into the adluminal compartment to continue their development (for reviews, see [1, 2]). Whereas this timely movement of developing germ cells across the BTB and the epithelium is essential to the completion of spermatogenesis, relatively few studies have investigated these events, let alone their regulation. Obviously, a thorough understanding of these events can yield new insights for the development of novel male contraceptives. For instance, an unexpected closing of BTB at the time when pre- and leptotene spermatocytes need to traverse the BTB will disrupt spermatogenesis, leading to infertility. Likewise, a prolonged opening of the BTB will also disrupt spermatogenesis, because germ cell antigens normally sequestered from the immune system will be exposed to the host immune system, which will mount an immunological attack on germ cells.

Recent studies from this laboratory have implicated that the events of germ cell movement are composed of intermittent phases of junction disassembly and reassembly interspaced by the protruding cytoplasmic processes of Sertoli cells that facilitate the physical translocation of germ cells from one site to another (for review, see [3]). An in vitro model to study the events of junction disassembly is not available yet, but Sertoli cells cultured in vitro are a useful model to examine the cascade of events leading to inter-Sertoli TJ assembly and reassembly. Using Sertoli cells cultured at 0.5–1.2 x 106 cells/cm2 on Matrigel-coated bicameral units or dishes, a transient but significant increase in the expression of ZO-1, a TJ-associated cytoplasmic protein, was detected at the time when inter-Sertoli TJs were being assembled [4–6]. These data suggest that ZO-1 may be involved in the assembly of inter-Sertoli TJs. Moreover, the expression of an array of molecules was induced coinciding with the assembly of inter-Sertoli cell junctions, which include N-cadherin, connexin 33, ß-catenin, prostaglandin D2 synthetase, myotubularin, and {alpha}2-macroglobulin [4, 5, 7–9]. However, the mechanism by which these molecules participate in the events of junction assembly is entirely unknown, though a recent study has demonstrated that transforming growth factor ß can regulate the timely expression of several TJ-associated proteins, such as occludin, ZO-1, and claudin-11, at the time of inter-Sertoli TJ assembly [6].

To date, several TJ-integral proteins have been identified, including occludin [10], the claudin multigene family [11–13], and junctional adhesion molecule [14]. Occludin is a 65-kDa protein localized at TJ strands [10, 15, 16]. It consists of four transmembrane domains, a long carboxyl-terminal cytoplasmic domain, a short N-terminal cytoplasmic domain, two extracellular loops, and one intracellular loop (for reviews, see [17–19]). These characteristics are well conserved among different mammalian species [20]. Among these domains, the first extracellular domain is rich in Tyr and Gly, which constitute approximately 60% of the amino acid residues, and is implicated in cell-cell coupling [20]. Introduction of an occludin construct into occludin-deficient fibroblasts enhanced cell adhesion; this adhesion could be inhibited by a synthetic peptide corresponding to the first extracellular loop [21]. On the other hand, addition of a synthetic peptide corresponding to the second extracellular domain of occludin to the Xenopus kidney epithelial A6 cell line disrupted the TJ permeability barrier, whereas a peptide corresponding to the first extracellular domain had no effects [22]. Taking these results together, it is apparent that the first extracellular loop is involved in cell-cell adhesion, whereas the second loop is important in the assembly and sealing of TJs. In the present study, we examined whether a 22-amino acid synthetic peptide corresponding to the second extracellular loop of rat occludin could affect the inter-Sertoli TJ permeability barrier in vitro and in vivo and, if it could, whether it could also reversibly disrupt spermatogenesis in vivo.

MATERIALS AND METHODS

Animals

Adult (250–300 g body weight [BW]) and 20-day-old Sprague-Dawley rats were obtained from Charles River Laboratories (Kingston, MA). All rats were housed at the Rockefeller University Laboratory Animal Research Center. These animals were maintained in accordance with the applicable portions of the Animal Welfare Act and the guidelines in the U.S. Department of Health and Human Services publication Guide for the Care and Use of Laboratory Animals. The use of animals for all studies described in this report was approved by the Rockefeller University Animal Care and Use Committee with protocol numbers 97117, 95129R, and 00111.

Preparation of Sertoli Cell Cultures

Primary Sertoli cells were isolated from 20-day-old Sprague-Dawley rat testes, and cell number was determined by a Coulter counter (Coulter Electronics, Hialeah, FL) as previously described [23]. Cell number was also confirmed by direct counting using a hematocytometer (Hausser Scientific, Horsham, PA) before cell plating so that the appropriate number of cells was adjusted in reference to the surface area of the culture dish (for a 12-well culture dish from Costar [Corning, Inc., Corning, NY], the surface area was ~3.79 cm2). Because freshly isolated Sertoli cells are in small aggregates of 5–15 cells, an aliquot of cell suspension was trypsinized (0.1% [w/v] trypsin in 1:1 [v/v] Ham F-12 Nutrient Mixture/Dulbecco modified Eagle medium [F12/DMEM]; Life Technologies, Inc., Gaithersburg, MD) for 1 min and then washed in F12/DMEM containing 1% soybean trypsin inhibitor (5 min, 800 x g), followed by two successive washes in F12/DMEM containing 5% fetal calf serum before counting on a hematocytometer. For low-cell-density cultures, Sertoli cells were plated at 5 x 104 cells/cm2 in 100-mm Petri dishes (~4.5 x 106 cells/100-mm dish per 9 ml of F12/DMEM); F12/DMEM was supplemented with 15 mM HEPES, 1.2 g/L of sodium bicarbonate, 10 µg/ml of bovine insulin, 5 µg/ml of human transferrin, 2.5 ng/ml of epidermal growth factor, 20 mg/L of gentamicin, and 10 µg/ml of bacitracin. Sertoli cells cultured at low cell density formed monolayers without the assembly of inter-Sertoli TJs when monitored by transepithelial electrical resistance (TER) measurement [24]. However, both adherens junctions (AJs) and gap junctions (GJs) were capable of forming [4]. For high-cell-density cultures, Sertoli cells were plated either on Matrigel (Collaborative Biochemical Products, Bedford, MA)-coated (diluted 1:7 [v/v] with F12/DMEM), 24-well dishes (effective surface area, ~1.88 cm2 with 2 ml of F12/DMEM per well) or on Matrigel-coated bicameral units (Millipore, Bedford, MA) (effective surface area was ~0.6 cm2) at a density of 0.6–3 x 106 cells/cm2 as previously described [24] to allow formation of TJs, AJs, and GJs, mimicking Sertoli cells found in vivo when assessed by various criteria [25]. Cell cultures were incubated in a humidified atmosphere of 95% air/5% CO2 (v/v) at 35°C, and TER readings were recorded 24 h later and designated as cultures at Day 1. These Sertoli cell cultures were shown to have a purity greater than 95% when examined microscopically [5, 25, 26].

Detection of Occludin Steady-State mRNA Level by Semiquantitative Reverse Transcription-Polymerase Chain Reaction

Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed essentially as described elsewhere [4, 24, 27, 28]. The RNA was extracted from cultured cells at specified time points by RNA STAT-60 (Tel Test "B," Inc., Friendswood, TX). Approximately 3 µg of total RNA were reverse-transcribed into cDNAs using 1 µg of oligo-dT15 and a Moloney murine leukemia virus reverse transcription kit (Promega, Madison, WI) in a final reaction volume of 25 µl. To quantify and compare the levels of occludin mRNA from various samples, occludin was coamplified with S16 so that the relative expression of occludin could be normalized against S16. In preliminary experiments, PCR was performed using different concentrations of template and primer pairs, and PCR products were examined over a range of 20–28 amplification cycles to ensure linearity of the target gene and S16. In most experiments, PCR was performed by combining 3 µl of the RT product with 0.4 µg each of occludin primers (sense primer: 5'-CTGTCTATGCTCGTCATCG-3', nucleotides 770–788; antisense primer: 5'-CATTCCCGATCTAATGACGC-3', nucleotides 1044–1063; Genbank accession no. AB016425), 80 ng each of rat ribosomal S16 primers (sense primer: 5'-TCCGGCAGTCCGTTCAAGTCTT-3', nucleotides 15–38; antisense primer: 5'-GCCAAACTTCTTGGATTCGCAGCG-3', nucleotides 376–399) [29], 5 µl of 10x PCR buffer, 3 µl of MgCl2 (25 mM), 8 µl of dNTPs (200 µM each of dATP, dGTP, dCTP, and dTTP), 2.5 U Taq DNA polymerase (Promega), and sterile, double-distilled water to a final volume of 50 µl. The cycling parameters for PCR were as follows: denaturation at 94°C for 1 min, annealing at 62°C for 2 min, and extension at 72°C for 3 min for a total of 23 cycles, followed by a 15-min extension at 72°C. To enhance the detection limit and to yield data for semiquantitative analysis following densitometric scanning of the resultant autoradiograms, PCR was performed in the presence of {gamma}-32P-labeled primer. Briefly, the sense primer of occludin and S16 were labeled at the 5'-end with [{gamma}-32P]dATP (specific activity, 6000 Ci/mmol; Amersham Pharmacia Biotech, Uppsala, Sweden) using T4 polynucleotide kinase (Promega). The relative ratio of the [{gamma}-32P]S16 (sense primer, ~10 000 cpm/PCR tube) to the [{gamma}-32P]occludin (sense primer) was the same as the unlabeled corresponding sense primer, so the resultant autoradiograms are the replicate of the ethidium bromide-stained gel. Approximately 10-µl aliquots of the PCR product were resolved onto 5% T polyacrylamide gels using 0.5x TBE (45 mM Tris-borate, 1 mM EDTA, pH 8.0) as a running buffer. The PCR products were visualized by ethidium bromide staining. Gels were then dried, and autoradiography was performed using Kodak X-OMAT AR film (Eastman Kodak, Rochester, NY). The resultant autoradiograms were densitometrically scanned at 600 nm using an UltroScan XL Enhanced Laser Densitometer (Amersham Pharmacia Biotech), and data were normalized against S16 to yield semiquantitative data.

Synthesis, Purification, and Characterization of the Occludin Synthetic Peptide

A 22-amino acid peptide corresponding to the second extracellular domain of rat occludin (NH2-GSQIYTICSQFYTPGGTGLYVD-COOH, amino acid residues 209–230; Genbank accession no. AB016425), which is furthest from the cell surface, and a 22-amino acid myotubularin (NH2-TKVNERYELCDTYPALLAVPAN-COOH, residues 156–177) [8] were obtained from SynPep Corp (Dublin, CA). These peptide sequences shared no homology to existing entries at GenBank using BLAST search software. However, the short stretch of sequence for rat occludin shared 90% homology among occludin isolated from different species. To purify the synthetic peptide, 500 µg of the crude peptide were dissolved in solvent A (5% acetonitrile/95% water, containing 0.1% [v/v] trifluoroacetic acid) and loaded onto a Vydac (Separations Group, Hesperia, CA) C18 reverse-phase high-performance liquid chromatography (HPLC) column (i.d., 4.6 x 250 mm) at a flow rate of 1 ml/min. The occludin peptide was then separated from other contaminants and eluted using a linear gradient of 15–65% solvent B (95% acetonitrile/5% water, containing 0.1% [v/v] trifluoroacetic acid) over a period of 30 min as described previously [8, 28]. The eluents were monitored by ultraviolet (UV) absorbance at 220 nm, and fractions of 0.5 ml each were collected. Fraction containing the occludin peptide was frozen, lyophilized, and further purified on a second C18 column. Thereafter, approximately 50 pmol of the purified occludin peptide were microsequenced to confirm its identity as previously described [28, 30, 31]. The repetitive yield was approximately 96%. The myotubularin synthetic peptide was subjected to the same purification and characterization scheme as the occludin peptide; results of these analyses from this laboratory have been described elsewhere [8].

Assessing the Integrity of the Inter-Sertoli TJ Permeability Barrier by Measuring the TER Across Sertoli Cell Epithelium

To assess the effects of occludin peptide on the assembly of inter-Sertoli TJs, Sertoli cells isolated from 20-day-old rat testes were cultured at 1.2 x 106 cells/cm2 to allow the assembly of inter-Sertoli TJs, and TER, which is a quantitative measurement of TJ integrity, across the Sertoli cell epithelium was quantified as previously described [5, 24]. Cells were plated on Matrigel (1:7)-coated HA (mixed cellulose esters) filters in the apical chamber (Millipore, Bedford, MA) [25, 28]. Great care was taken so that air bubbles were not trapped between Sertoli cell aggregates, which is the major obstacle to obtaining steady TER across the Sertoli cell epithelium, because air bubbles create physical pores between adjacent Sertoli cells. The TER across the Sertoli cell epithelium at specific time points was determined by a Millicell electrical resistance system (Millipore) as described elsewhere [24, 32]. The resistance in ohms was multiplied by the effective surface area of the bicameral unit (~0.6 cm2) to yield the areal resistance (ohm·cm2). The net value of electrical resistance was then computed by subtracting the background, which was measured on the Matrigel-coated, cell-free chambers, from values of Sertoli cell-plated chambers. To minimize temperature-induced fluctuations during TER measurement, cultures were stabilized at room temperature for 20–30 min before recording TER across the Sertoli cell epithelium. Synthetic occludin peptide at 0.2–4 µM was included in both the basal (0.5 ml of F12/DMEM containing 0.03% [v/v] dimethyl sulfoxide [DMSO]; DMSO was used to solubilize the peptide in medium) and apical (0.5 ml of F12/DME containing 0.03% [v/v] DMSO) chambers of the bicameral units 24 h after freshly isolated Sertoli cells were plated onto Matrigel-coated units (Day 1). Peptide was included in F12/DMEM containing 0.03% (v/v) DMSO when media were replaced daily. In selected experiments, synthetic occludin peptide was removed from the Sertoli cell epithelium by rinsing cells with two successive washes of F12/DMEM without peptide, and subsequent media also contained no peptide. Control experiments included: 1) Sertoli cells cultured alone, 2) Sertoli cells cultured with vehicle only (media with 0.03% [v/v] DMSO), and 3) Sertoli cells cultured in the presence of 4 µM of the 22-amino acid synthetic myotubularin peptide as described above. Each time point contained triplicate cultures, and each experiment was repeated two or three times using different batches of Sertoli cells. We have selected TER measurement to quantify the assembly and maintenance of inter-Sertoli TJs as opposed to other methodologies, which include: 1) restriction of diffusion of [3H]inulin, [125I]-BSA, or fluorescein isothiocyanate-labeled dextran across the Sertoli cell epithelium; 2) maintenance of nonequilibrium of the media in the apical and basal chamber of the bicameral units; and 3) polarized secretion of Sertoli cell products, such as transferrin, rABP (rat androgen binding protein), testin, clusterin, and {alpha}2-macroglobulin, as described elsewhere [25, 26], for the following reasons. First, this technique is widely adopted by cell biologists in the field [22, 33]. Second, it yields quantitative measurement on the assembly and maintenance of inter-Sertoli TJs. Third, and most important, results obtained by TER measurement are consistent with those of other tedious approaches as described above, such as restriction diffusion of [3H]inulin and fluorescein isothiocyanate-labeled dextran monitored by a Tecan GENios cytofluorometer (Salzburg, Austria).

Intratesticular Injection of Occludin Peptide and Histological Analysis of the Testis

To assess the in vivo effects of the occludin peptide on spermatogenesis, peptide was administered to testes of adult rats by direct intratesticular injection. Peptide was suspended in 0.9% sterile saline and then sterilized by exposure to UV radiation for 5 min. It was noted that this brief UV treatment to sterilize the peptide suspension before its use did not alter its structure, and this was verified by two approaches. First, UV-treated peptide retained the same retention time on reverse-phase HPLC using the Vydac C18 column (i.d., 4.6 x 250 mm) [30, 31] when compared to peptide before the UV treatment. Second, the primary sequence of the UV-treated peptide remained unaltered when direct protein microsequencing was performed as described elsewhere [28, 30, 31]. Adult rats between 250 and 300 g BW were anesthetized with Metofane (2,2-dichloro-1,1-difluoroethyl methyl ether; Mallinckrodt Veterinary, Inc., Mundelein, IL) before treatment. Rats received either 300 µl of 0.9% sterile saline (vehicle control), no treatment (control), or 1.5–10 mg of occludin peptide suspended in 300 µl of 0.9% sterile saline intratesticularly. The right testis of each animal received the peptide or vehicle, and the left testis of the same animal was not treated and used as a control. Peptide or vehicle was administered at three sites per testis, with an approximately 100-µl sample per site using a 26-gauge needle (Becton Dickinson, Rutherford, NJ) essentially as previously described [24, 34]. In another control group, rats were injected with a synthetic 22-amino acid peptide of NH2-TKVNERYELCDTYPALLAVPAN-COOH based on a known Sertoli cell protein, rat myotubularin (rMTM), under investigation in this laboratory [8, 9] and that has no sequence homology with occludin. Three rats were used for each time point in each treatment group, and rats were killed by CO2 asphyxiation at specific time points. Testes were removed immediately and fixed in 10% neutral buffered formalin. Testes were embedded in paraffin and dehydrated in graded ethanol. For morphological analysis, 5-µm sections were cut and stained with hematoxylin and eosin. Approximately 50 sections were examined at different sites for each testis using an Olympus BX40 microscope (Olympus, Tokyo, Japan) interfaced to an Olympus PM-30 Exposure Control Unit.

Assessing the Occludin-Peptide Induced Disruption of the BTB by Micropuncture Techniques

Radioiodination of BSA Briefly, 5 µg of BSA (RIA grade, 68 kDa; Sigma, St. Louis, MO) was radioiodinated by Iodogen [35] using 1 mCi of [125I]-sodium iodide (Amersham Pharmacia Biotech) as described elsewhere [36].

Detection of [125I]-BSA in seminiferous tubular fluid and rete testis fluid At 2, 4, 6, and 12 wk after intratesticular administration of 1.5 mg of either occludin or myotubularin peptide per testis as described above, administered at three sites to the right testis with the left testis of the same animal being used as a control, rats (n = 4–6 per time point, ~250 g BW at the time of peptide treatment) were anesthetized with ketamine HCl (Fort Dodge Laboratories, Inc., Fort Dodge, IA) at 60 mg/kg BW. Micropuncture was performed essentially as described elsewhere [37, 38]. Briefly, testes were exposed through an abdominal incision, and the efferent ducts were ligated with surgical silk thread. Testes were then returned to the scrotum. The wound was cleansed with 70% ethanol, surgically closed, and the animals allowed to recover. Twenty-four hours after efferent duct ligation, rats were anesthetized by ketamine HCl. Bilateral nephrectomy was then performed to prevent renal excretion of [125I]-BSA, and approximately 6 x 106 cpm of [125I]-BSA was infused into the rat via the jugular vein. Two hours after infusion, testes were removed, and rete testis fluid (RTF) and seminiferous tubular fluid (STF) were collected as described previously [37, 38] for radioactivity determination in a {gamma}-counter. The left testis from the same animal, which did not receive either the occludin or myotubularin peptide, served as a control, and both STF and RTF were also collected from this testis for radioactivity determination to assess the integrity of the BTB.

Statistical Analysis

Results were analyzed for statistical significance either by Student t-test to compare treated samples with their corresponding controls or by ANOVA using the GB-STAT Statistical Analysis Package (version 7.0; Dynamic Microsystems, Inc., Silver Spring, MD). Using the Tukey honestly significant difference (HSD) test for ANOVA, results of individual samples were compared to controls and to samples subjected to the same treatment within the same group. In all culture experiments studying cellular gene expression or for TER measurement to assess inter-Sertoli TJ permeability barrier, each time point had replicate cultures, and each experiment was repeated two or three times using different batches of Sertoli cells.

RESULTS

Expression of Occludin by Sertoli Cells Correlates with Assembly of the Inter-Sertoli TJ Permeability Barrier In Vitro

When Sertoli cells were cultured at different cell densities ranging between 2 x 104 and 3 x 106 cells/cm2 on Matrigel-coated bicameral units, a steady increase in TER across the Sertoli cell epithelium was noted (Fig. 1). The assembly of inter-Sertoli TJs was completed by Day 4, as manifested by a stable TER across the Sertoli cell epithelium (Fig. 1). These results were consistent with those obtained using other techniques to assess the inter-Sertoli TJ permeability barrier, such as the restricted diffusion of [3H]inulin across the Sertoli cell epithelium and polarized secretion of Sertoli cell secretory products, such as rABP, transferrin, and testin [25, 39, 40]. Because TER measurement yields a quantitative assessment on the inter-Sertoli TJ assembly, it does not require the use of radioactive isotopes, is highly reproducible, is relatively easy to set up and maintain, and is widely used by cell biologists in the field [22, 33]. We thus selected this method as opposed to other approaches.



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  		FIG. 1. Assembly of the inter-Sertoli TJ permeability barrier in vitro is dependent on the Sertoli cell numbers. The TER across the Sertoli cell epithelium was quantified using Sertoli cells cultured at different densities to assess the integrity of the TJ barrier. Each data point is the mean ± SD of nine determinations from three separate experiments. Each experiment had triplicate cultures

Sertoli cells cultured on Matrigel-coated bicameral units at different cell densities displayed a similar TER pattern over time in culture during TJ assembly, but the "tightness" of the inter-Sertoli TJ positively correlated with the cell density. When Sertoli cells were cultured at 3 x 106 cells/cm2, a mild decline in TER was noted after Day 4 in three different experiments. The explanation for this observation is not immediately known. It is possibly a result of cell overcrowding and death, accumulation of metabolic wastes, and insufficient nutrient flow. This postulate is supported by morphological studies showing that cells cultured at this high density are accompanied by an increase in DNA fragmentation derived from degenerating Sertoli cells [5]. At low cell density (2 x 104 cells/cm2), no measurable TJ permeability was detected, possibly due to the lack of close cell proximity to allow TJ assembly because of insufficient cell number (Fig. 1). At high cell density (1.2 x 106 cells/cm2), a significant but transient increase was found in occludin steady-state mRNA level between Days 2 and 4.5 (Fig. 2, A and B) at the time when inter-Sertoli TJs were assembled (compare Figs. 1 and 2), illustrating that TJ assembly required de novo synthesis of occludin, which was one of the building blocks of the TJs. After Day 5, the steady-state occludin mRNA returned to the basal level, similar to that of Day 1 (Fig. 2, A and B). Such a transient induction in occludin expression was not detected in low-cell-density cultures at 2 x 104 cells/cm2 (compare Fig. 2C with Fig. 2, A and B). These results were consistent with previous observations showing a correlation between the induction of ZO-1, which is also a TJ-associated protein, and inter-Sertoli TJ assembly [4, 5].



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  		FIG. 2. Changes of the steady-state occludin mRNA level during the assembly of inter-Sertoli TJs in vitro. Sertoli cells were cultured at a density of 1.2 x 106 cells/cm2 (A and B) and 2 x 104 cells/cm2 (C). Cells were terminated at specified time points between Day 1 (i.e., 24 h after their isolation) and Day 8 for total RNA extraction. Semiquantitative RT-PCR was used to detect the changes of occludin expression in these samples. Coamplification was performed using rat ribosomal S16. The RT-PCR products were resolved onto 5% T polyacrylamide gels (A and C). In B, the corresponding densitometric scanned results using blots such as the one in A are shown. Densitometric scanning was performed at 600 nm, and data were normalized against S16. Each bar represents the mean ± SD of two separate experiments. Each experiment had replicate cultures. ns, Not significantly different by ANOVA. *Significantly different by ANOVA (P < 0.01)

Reversible Perturbation of Inter-Sertoli TJ Permeability Barrier In Vitro by Use of a 22-Amino Acid Synthetic Peptide Corresponding to a Segment of the Second External Loop of Occludin

A 22-amino acid synthetic peptide corresponding to the outermost region of the second external loop of rat occludin was assessed for its ability to affect the assembly of inter-Sertoli TJs. Following its synthesis, the synthetic occludin peptide was purified by reverse-phase HPLC (Fig. 3, A and B), and its identity was confirmed by direct protein microsequencing. Addition of occludin peptide to the Sertoli cell epithelium 24 h after isolation at a density of 1.2 x 106 cells/cm2 induced a dose-dependent decline in TER (Fig. 4A). This peptide-induced disruption of the paracellular permeability barrier could be reversed after its removal from the culture (Fig. 4B). Sertoli cells incubated with occludin peptide at 4 µM caused a significant decline in TER, which was approximately 50% when compared to untreated controls on Days 4–5. In selected experiments, when the occludin peptide was removed on Day 5 from the bicameral units by two successive washes using F12/DMEM, the inter-Sertoli TJ permeability barrier could be reassembled, making the TER reading indistinguishable from that of controls within 3–4 days (Fig. 4B). Interestingly, the time that it took to reassemble the disrupted inter-Sertoli TJ induced by the occludin peptide was roughly equivalent to that of the inter-Sertoli TJ assembly using freshly isolated Sertoli cells in vitro, which was different from the Ca2+ depletion-induced TJ leakiness, because it took the Sertoli cell only 90 min to reseal disrupted TJs [24]. The effects of occludin peptide in perturbing the inter-Sertoli TJ permeability barrier were reversible after the peptide removal, so it was apparent that the occludin peptide was nontoxic to the Sertoli cells. When a 22-amino acid rMTM peptide at 4 µM was used instead of the occludin peptide, it had no apparent effects in perturbing the inter-Sertoli TJ barrier (Fig. 4B). Also, when the cell viability in the occludin peptide-treated cultures was assessed by trypan blue dye-exclusion test versus control cultures in selected experiments, no apparent differences were detected (data not shown).



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  		FIG. 3. Purification of the occludin peptide by HPLC and microsequencing for identity confirmation. A) Purification of the synthetic occludin peptide. Briefly, approximately 500 µg of the crude occludin peptide was loaded onto a Vydac C18 reverse-phase column (i.d., 4.6 x 250 mm) as described in Materials and Methods. A total of 10 peptide peaks were detected when eluents were monitored by UV absorbance at 220 nm. The purified occludin peptide eluted in fraction 27 under protein peak 6, as denoted by the solid bar. B) About 1/5 of the sample (i.e., fraction 27 under peak 6) obtained in A was further purified on a second Vydac C18 reverse-phase HPLC column. Purified occludin peptide was eluted in fraction 32 under peak 2. Direct protein microsequencing of the purified occludin peptide was performed as described in Materials and Methods, which identified 13 amino acids from its N-terminus. The letter X represents an amino acid that could not be assigned unequivocally



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  		FIG. 4. Effect of a 22-amino acid occludin peptide corresponding to the second extracellular loop of rat occludin (residues 209–230) on the inter-Sertoli TJ permeability barrier in vitro. A) Dose-dependent effects of occludin peptide on the inter-Sertoli permeability barrier quantified by TER measurement. Sertoli cells were cultured on Matrigel-coated bicameral units (effective culture area, ~0.6 cm2) as described in Materials and Methods. Occludin synthetic peptide at 0.2, 0.4, and 4 µM was added to both the apical and the basal compartment of the bicameral unit 24 h after cell plating. As a vehicle control, DMSO at 0.03% was used. Media containing corresponding peptide at specified concentrations were replenished every 24 h in both apical and basal compartments. Each time point consists of triplicate cultures in each experiment. B) Reversible effect of the occludin peptide on the inter-Sertoli permeability barrier. In one set of experiments, the peptide (0.4 µM) was removed from cell cultures by two successive washes with F12/DMEM, and TER across the Sertoli cell epithelium was then quantified at specific time points. The myotubularin peptide (4 µM) had no apparent effects in perturbing the TJ permeability barrier. Each time point is the mean ± SD of nine determinations from three separate experiments

Reversible Effects of Occludin Peptide on Testicular Weight, Testicular Size, and Spermatogenesis In Vivo

Effects on testicular weight and size Following HPLC purification, occludin or myotubularin peptide was suspended in 0.9% sterile saline and sterilized under UV radiation for 5 min. Each rat in the experimental group received an intratesticular injection of 300 µl of saline containing 1.5 mg of the corresponding purified peptide in the right testis. The peptide suspension was distributed in each testis at three sites (~100 µl/site) as described in Materials and Methods and detailed elsewhere [24, 34]. Intratesticular injection of this purified occludin peptide (1.5 mg/testis) caused a reduction in testicular weight (right testis) within 2 wk when compared to control rats that received no treatment, vehicle (0.9% sterile saline) alone, or myotubularin peptide (Fig. 5A). By 4 wk, the right testis was approximately 40% of the control (left testis) by weight (Fig. 5A), and the myotubularin-treated testis was not different from the untreated control testis for all time points examined, both by weight (Fig. 5A) and by size (data not shown). Whereas an occludin peptide-induced decline in testicular weight (Fig. 5A) and testicular size (Fig. 5B) was noted, the appearance of the testis and the epididymis appeared normal. By 6–9 wk, the occludin peptide-treated testis (right testis) was approximately 70% by weight of the control (left testis). In another set of experiments, when 10 mg of occludin peptide were injected to the right testis, changes in the testicular weight (data not shown) were similar to those in rats receiving 1.5 mg of peptide, which seemingly suggests that a lower effective dose to perturb spermatogenesis could be used.



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  		FIG. 5. Changes in testicular weight and size after intratesticular injection of occludin or myotubularin peptide. A) The right testis of an adult rat received an intratesticular injection of 1.5 mg of occludin peptide, and the left testis received either no treatment, rMTM, or vehicle alone. At 2, 4, 6, and 9 wk (n = 2 per time point) after the synthetic occludin or rMTM peptide injection, rats were killed, and the weights of both testes were determined. Control rats received no treatment, and the testicular weight (left testis only) in this group at 2, 4, 6, and 9 wk were 1.6, 1.7, 1.8, and 1.9 g, respectively. Results are means of two testes from two rats. B) Changes in testicular sizes in testis pair by 27 and 58 days after rats received an intratesticular injection of 1.5 mg of occludin peptide per testis (i.e., 3 mg of peptide/testis pair) when compared to a control testis pair receiving no treatment. ns, Not significantly different from control rats by Student t-test; Ocdn, occludin; rMTM, rat myotubularin. *Significantly different from control rats by Student t-test (P < 0.01). Scale in millimeters

Effects on spermatogenesis Figure 6, A–C, shows the control rat testes receiving either no treatment (Fig. 6A), at 14-day post-intratesticular injection of vehicle alone (Fig. 6B), or at 14-day post-intratesticular injection of saline (Fig. 6C). Figure 6, D–F, shows another control set of testes in which rats received an intratesticular injection of the myotubularin peptide at 1.5 mg/testis at 10 (Fig. 6D), 23 (Fig. 6E), and 60 days (Fig. 6F) posttreatment. Morphological analysis of the treated testis revealed that more advanced germ cells, such as elongated spermatids, began to deplete from the epithelium between 8 (Fig. 6G) and 16 days after the occludin peptide treatment. Massive depletion of germ cells from the epithelium in virtually all tubules examined was noted by 27 days after intratesticular occludin peptide injection (Fig. 6, I and J). In addition, the seminiferous tubules of the occludin-treated testes shrunk significantly, with the tubular diameter reduced by as much as 20–30% when compared to control rats or testes receiving vehicle only or myotubularin peptide alone (compare Fig. 6, I and J, to Fig. 6, A–C). Germ cells began to repopulate the epithelium after 27 days post-occludin peptide treatment. By 47 days, spermatocytes were clearly visible in all tubules examined (Fig. 6K), and the morphology of the seminiferous epithelium appeared indistinguishable from that in control rats by 68 days post-occludin peptide treatment (Fig. 6L), showing full recovery from the occludin peptide-induced damage in the testes (Fig. 6L). That the testes recovered almost fully within 40 days (compare Fig. 6L at 68 days to Fig. 6, I and J, at 27 days posttreatment) suggests that spermatogonia were not destroyed by the occludin peptide treatment.



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  		FIG. 6. Antispermatogenic effects of the 22-amino acid synthetic occludin peptide following its administration to adult rats via intratesticular injection. Adult rats weighing between 250 and 300 g BW received no injection (control) or were treated with vehicle (0.9% sterile saline or 5% DMSO/6% ethanol in 0.9% saline), purified rMTM peptide (NH2-TKVNERYELCDTYPALLAVPAN-COOH), or purified synthetic occludin peptide. Peptide-treated and control rats were killed at specified time points. Testes were removed and processed for histological analysis as described in Materials and Methods. Approximately 50 different cross-sections of the testes from different rats within the same group were examined, and representative micrographs were shown. A) Control rat testis that received no treatment. B) Fourteen days after an intratesticular injection with vehicle (0.9% sterile saline or 5% DMSO/6% ethanol in 0.9% saline at three sites). C) Fourteen days after an intratesticular injection with 0.9% sterile saline at three sites. D–F) Cross-sections of rat testes at 10 (D), 23 (E), and 60 (F) days after receiving a single intratesticular injection of the rMTM peptide at 1.5 mg/testis (at three sites, see Materials and Methods). When these micrographs were compared to the control testes in A–C, no morphological changes were observed. G–L) Rats received an intratesticular injection of purified occludin peptide at 1.5 mg/testis at three sites, and the testis was removed for histological analysis at 8 (G), 16 (H), 27 (I and J), 47 (K), or 68 (L) days thereafter. It was noted that round and elongated spermatids began to deplete from the seminiferous epithelium 16 days after the peptide treatment (H vs. A–C), and virtually all the spermatids were depleted from the epithelium by 27 days (I and J vs. A–C). Germ cells, largely spermatocytes, began to repopulate the epithelium 47 days posttreatment, and by 68 days, the morphology of the tubule was almost indistinguishable from that in control rats (K and L vs. A–C), illustrating that this treatment is completely reversible. Ocdn, Occludin synthetic peptide; rMTM, rat myotubularin peptide. Bar = 120 µm

Effects of Occludin Peptide on the BTB

To investigate whether the occludin peptide treatment could specifically affect the BTB's functionality, the integrity of the BTB was assessed following the intratesticular injection of either occludin or myotubularin peptide. Peptide was administered at three sites per testis, and the other testis of the same rat (~270–300 g BW) was used as the control (n = 4–6 rats per time point). Results shown in Figure 7 clearly illustrate a disruption of the BTB following an intratesticular injection of occludin peptide at 1.5 mg/testis. An accumulation of [125I]-BSA was noted in the STF (Fig. 7A) and RTF (Fig. 7B) in the occludin peptide-injected testes between 2 and 6 wk posttreatment compared to the untreated testes in the same rats after infusion of [125I]-BSA through the jugular vein. The peptide-induced damage to the BTB appeared to be reversible, because a drastic decline was noted in [125I]-BSA accumulation in both STF and RTF (Fig. 7, A and B) by 12 wk posttreatment, coinciding with the recovery of the seminiferous epithelium when examined by histological analysis (data not shown) similar to those shown in Figure 6. Moreover, the level of radioactivity in STF and RTF by 12 wk collected from peptide-treated rats became indistinguishable from that in control testes, which had not been exposed to the occludin peptide (Fig. 7). This occludin peptide-induced damage to the BTB appears to be specific, because the rat myotubularin peptide failed to induce disruption of the BTB as the radioactivity detected in either the STF or RTF in myotubularin peptide-treated rats at all time points was indistinguishable from that in control rats (Fig. 7, A and B).



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  		FIG. 7. Reversible disruption of the BTB following an intratesticular injection of the synthetic occludin or myotubularin peptide. A dose of 1.5 mg of purified synthetic occludin or myotubularin peptide was injected into the right testis of an adult rat, and the left testis served as a control (n = 4–6 rats for each time point). At 2, 4, 6, and 12 wk after the intratesticular peptide injection, efferent duct ligation was performed. Twenty-four hours later, bilateral nephrectomy was performed, and approximately 6 x 106 cpm of [125I]-BSA suspended in 10 µl of PBS buffer were infused into each rat via the jugular vein. The STF (A) and RTF (B) were collected 2 h thereafter, as described in Materials and Methods, and [125I]-BSA was quantified by a {gamma}-counter. Approximately 10 and 50 µl of RTF and STF, respectively, were routinely obtained from each testis. Data were normalized against either testicular weight for STF or per milliliter of fluid for RTF. ns, Not significantly different from control by Student t-test; *significantly different from control by Student t-test (P < 0.01)

DISCUSSION

The inter-Sertoli TJs that create the BTB play an important role in the testis. First, the BTB (and, therefore, TJs) serves as a fence between the seminiferous epithelium and the basal lamina, restricting the paracellular transport of molecules. Second, TJs constitute the major part of the BTB that segregates germ cell development from the systemic circulation, creating a favorable milieu for spermatogenesis [41]. Third, TJs create and maintain cell polarity (for reviews, see [42–44]). Several TJ-associated proteins, such as ZO-1 [45, 46], cingulin [42], occludin [47], and claudin-1, -3, -4, -5, -7, -8, and -11 (for reviews, see [18, 48]) have been found in the testis. Among these proteins, only ZO-1, a cytoplasmic protein, has been extensively studied in the testis [4, 5, 45, 46]. At least nine integral TJ-proteins are found in the TJ strands of the testis, which, in turn, constitute the inter-Sertoli TJs and the BTB. These include occludin [10]; occludin 1B, which is a variant of occludin containing an additional 193-base pair insertion with a unique N-terminal sequence [49]; and claudin-1, -3, -4, -5, -7, -8, and -11 [11–13]. Approximately 24 claudin species have been identified in different TJs, and their distribution varies in different organs [11–13, 50]. It was reported that occludin was concentrated in TJs of mouse/rat but not human/guinea pig Sertoli cells [47]. Studies by immunohistochemistry have shown that occludin is expressed by Sertoli cells in the mouse during early embryonic and postnatal development and is localized at the site of inter-Sertoli TJs by Postnatal Day 14, at the time of BTB assembly [51], suggesting its involvement in BTB assembly at puberty. More recent studies investigating the regulation of intestinal TJs have shown that zonulin is a potential regulator of TJ dynamics; is found in the TJ of fetal brain, heart, and intestine; and becomes highly concentrated in adult heart and intestine (but not brain). However, its presence in the testis is not yet known, and its cDNA has not been cloned [52, 53].

In this report, we have shown that occludin is a marker to monitor inter-Sertoli TJ assembly in vitro. An induction in occludin expression is detected at the time of TJ assembly, suggesting that occludin is required for TJ formation. Both the expression of ZO-1 and occludin are induced when inter-Sertoli TJs are being assembled, which is consistent with the biochemical findings that the cytoplasmic domain of occludin being associated with ZO-1 at a stoichiometric ratio of 1:1 [47]. These results are also consistent with the postulate that ZO-1 acts as a linker to bridge an integral membrane TJ protein, such as occludin, and the actin-based cytoskeleton during TJ biogenesis [54, 55]. The timely induction of occludin expression during TJ assembly is also consistent with several reports of an elevated level of phosphorylated occludin during TJ assembly [56, 57]. Functional analysis of occludin in different epithelial systems has shown that occludin plays a crucial role in the assembly of TJs (for reviews, see [17, 58, 59]). For instance, an increase in TER was detected in MDCK (Madin-Darby canine kidney) cells following transfection with a full-length occludin cDNA [33, 60]. However, occludin-deficient embryonic stem cells are capable of differentiating into polarized epithelial cells bearing TJs [61], suggesting that claudins (another TJ-integral transmembrane protein family, of which at least 24 members have been identified to date) (for reviews, see [18, 19, 48]) or other, yet-to-be identified TJ integral proteins could supersede the role of occludin in TJ assembly, which may also associate with the underlying cytoplasmic TJ protein, ZO-1. For instance, other studies have shown that claudin-1, -3, -4, -5, -7, -8, and -11 are present in the testis, with claudin-11 being restricted to the brain, kidney, and the testis, suggesting that epithelial cells can utilize other members of the claudin family to construct the needed TJs (for review, see [19]). Furthermore, addition of a 44-amino acid synthetic occludin peptide corresponding to the entire second external domain of chick occludin in the Xenopus kidney epithelial cell line A6 can cause a drastic reduction of TER and disruption of paracellular permeability as assessed by paracellular flux assay [22]. Likewise, a 22-amino acid peptide corresponding to this second extracellular loop of occludin, the region furthest away from the cell surface, can also perturb the inter-Sertoli TJ permeability barrier dose-dependently, as demonstrated in this report. These results thus suggest that this region of occludin, which is furthest away from the cell surface, confers to the TJ functionality. Because removal of the peptide from media of treated Sertoli cells allows resealing of the TJ permeability barrier and use of a 22-amino acid synthetic peptide based on a stretch of sequence in myotubularin, another Sertoli cell protein, failed to perturb the inter-Sertoli TJ permeability barrier in vitro, these observations illustrate the specificity of the treatment.

These findings clearly demonstrate that the assembly of inter-Sertoli TJs is a dynamic event requiring de novo synthesis, targeting, and assortment of TJ-associated proteins to the site of TJs. The occludin peptide-induced disruption in the TJ barrier is possibly mediated by one of the following mechanisms. First, it might be possible that Sertoli cells were using these peptides as building blocks for TJ assembly. However, because they did not have the structural confirmation of the entire molecule to reinforce the TJ functionality, TJs became perturbed and disrupted. Second, homotypic interactions of the synthetic peptides with other intact occludin molecules between two neighboring Sertoli cells caused the recruitment of intact and functional occludin to the same site to become impossible. Thus, the TJ permeability barrier became disrupted. Several reports have, indeed, demonstrated the homotypic interactions of occludin between adjacent cells. For instance, fibroblasts (non-TJ-bearing cells), when transfected with occludin, exhibited cell adhesion activity [21]. Studies by immunoprecipitation have shown that exogenous occludin bound to endogenous Xenopus occludin in vivo, illustrating occludin that oligomerized during TJ assembly [62]. In addition, overexpression of occludin in Sf9 cells induced the formation of multilamellar structures with TJ-like strands, possibly via the polymerization of multiple occludin molecules within the lipid bilayers [63].

Apparently, the effects of occludin peptide on the inter-Sertoli TJ permeability barrier in vitro, as shown in this study, were not the result of cell toxicity. First, the effect of the occludin peptide was reversible following its removal, reflecting that cells were indeed viable. Second, this effect was specific to the occludin peptide, because use of a 22-amino acid rat myotubularin peptide failed to affect the assembly of inter-Sertoli TJs. Several reports in the literature described investigations utilizing synthetic occludin peptides to study their effect in vitro. First, a recent study used synthetic peptides corresponding to the first external loop of occludin to examine their role in cell-cell adhesion. When occludin-transfected fibroblasts were incubated with peptide corresponding to the first external loop of occludin, this peptide inhibited occludin-induced cell adhesion [21], demonstrating that the first external loop may be responsible for cell-cell adhesion. Interestingly, addition of peptide, which is homologous to the first external loop of chick occludin, to Xenopus A6 cell cultures also prevented the resealing of TJs, whereas a 10-amino acid peptide corresponding to the second extracellular domain had no effect [64]. However, Wong and Gumbiner [22] have demonstrated that a peptide synthesized based on the entire first external loop of chick occludin (a 44-amino acid peptide) failed to disrupt TJs in cultured Xenopus A6 cells, but that a 44-amino acid peptide corresponding to the entire second external loop did perturb TJ assembly. The reason for such experimental discrepancies is not immediately known. However, it is possible that they result from differences in the selection of stretches of amino acid sequences for peptide design by these investigators. For instance, the 22-amino acid peptide used in our study that could perturb inter-Sertoli TJ assembly covers the outermost region of the second external loop, which is farthest away from the cell surface, whereas the 10-amino acid peptide used by Lacaz-Vieira et al. [64], which failed to inhibit TJ assembly, corresponds to the descending portion of the second external loop, which is closer to the cell surface. Wong and Gumbiner [22] used a 44-amino acid peptide that could also perturb TJ assembly and that covered the entire second external loop.

For the past several decades, development of new male contraceptives has largely focused on manipulating the hypothalamus-pituitary-testicular axis to disrupt spermatogenesis (for review, see [65]). Administration of either high doses of testosterone or a combination of testosterone and synthetic progestins can inhibit pituitary gonadotrophin secretion, which, in turn, leads to oligospermia or azoospermia [66]. This inhibitory effect on spermatogenesis is reversible, but the exogenous administration of steroids or polypeptide hormones can interfere with the hormonal balance and, possibly, induce undesirable effects. As shown in the present study, intratesticular injection of occludin peptide can induce reversible aspermatogenesis. The possible mechanism of the occludin peptide-induced germ cell loss may be the result of BTB disruption, as demonstrated in the present study. Such damage to BTB may lead to an influx of immune cells into seminiferous epithelium, which then mount an attack to the autoantigenic germ cells that normally are sequestered from the systemic circulation. Because TJs closely associate with AJs functionally, spatially, and biochemically, disruption of TJs would also induce AJ dissociation, which, in turn, would induce detachment of germ cells from the epithelium. The effects of the occludin peptide-induced changes in the testis appear to be specific, because the myotubularin peptide failed to induce similar effects on spermatogenesis. As such, the use of an occludin peptide or other peptide-based reagents homologous to selected TJ or AJ proteins to impair spermatogenesis may provide a potential approach to arrest spermatogenesis. However, the major drawback of such an approach for male contraception is the requirement of an intratesticular injection, which is highly uncomfortable. Also, the disruption of the BTB, though reversible, is also uncomfortable to the treated animals and can possibly cause pain to those animals. Work is now in progress to conjugate this occludin peptide to a recombinant, modified FSH in which the biological potency is eliminated but the FSH receptor-binding activity is retained as an alternative delivery system.

In summary, occludin is a useful marker to monitor inter-Sertoli TJ assembly in vitro. Its second external loop, in particular the outermost region of the loop, apparently is important to confer to the TJ functionality and the inter-Sertoli TJ permeability barrier. More important, the in vitro effects of this synthetic occludin peptide can be reproduced in vivo since a synthetic peptide based on the second extracellular loop farthest away from the cell surface can induce aspermatogenesis when administered in vivo.

NOTE ADDED IN PROOF

Sequence alignment of the entire second extracellular loop of occludin among five different species ([19, 20, 59]; GenBank Accession Numbers: AB016425, NP-032782, AAC50451, A49467). This spans from residues 199 to 243 in the rat versus 187 to 227 in the chicken. For rat occludin, the underlined stretch of amino acid sequence represents the 22-amino acid peptide that has been synthesized, which corresponds to the outermost region of the second extracellular loop and was used in the present study.


FOOTNOTES

First decision: 17 April 2001.

1 Supported in part by grants from the Noopolis Foundation (to C.Y.C.), National Institutes of Health (U54-HD-13541-20S to C.Y.C.), the CONRAD Program (CICCR CIG 96-05A to C.Y.C.), and the Hong Kong Research Grant Council (HKU 7245/00M to W.M.L. and C.Y.C.). Back

2 Correspondence: C. Yan Cheng, Population Council, 1230 York Avenue, New York, NY 10021. FAX: 212 327 8733; y-cheng{at}popcbr.rockefeller.edu Back

Accepted: June 8, 2001.

Received: March 22, 2001.

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