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Acute Adverse Effects of the Indenopyridine CDB-4022 on the Ultrastructure of Sertoli Cells, Spermatocytes, and Spermatids in Rat Testes: Comparison to the Known Sertoli Cell Toxicant Di-n-pentylphthalate (DPP)

MICHAEL J. DYKSTRA

PETER C. MANN

GARY R. MARSHALL

From ^* BIOQUAL Inc, Rockville, Maryland; Laboratory for Advanced Electron and Light Optical Methods, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina; Experimental Pathology Laboratories Inc, Herndon, Virginia; and Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.

Correspondence to: Dr Sheri Ann Hild, BIOQUAL Inc, 9600 Medical Center Dr, Rockville, MD 20850 (e-mail: shild{at}bioqual.com).

Received for publication November 28, 2006; accepted for publication March 22, 2007.

	Abstract

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Acute effects of CDB-4022 on testicular ultrastructure were determined. Rats were treated orally with vehicle or a maximally effective single dose of CDB-4022 or Di-n-pentylphthalate (DPP). Preserved testes were processed for transmission electron microscopy. Sertoli and germ cells of vehicle-treated rats demonstrated normal morphological characteristics. Disruption of Sertoli cell ultrastructure was apparent in CDB-4022-treated rats by 3 hours. A decrease in the presence of nucleoli, an increase in the amount and diameter of swollen smooth endoplasmic reticulum, and decreases in cytoplasmic ground substance were observed. The severity of these degenerative effects increased at 6 and 12 hours: Vacuoles were apparent; increased cellular debris, swollen mitochondria, and phagocytic structures were observed; and membranes became more disorganized. Similar ultrastructural changes were observed in the Sertoli cells of DPP-treated rats. By 3 hours, spermatocytes and spermatids were adversely affected by CDB-4022 treatment with swelling of the nuclear envelope. The Step 8 spermatids were especially noteworthy; chromatin was more diffuse and rarefied, the nuclear envelopes were incomplete or broken, and the position of the spermatid nucleus within the cell and relative to Sertoli cell cytoplasm was unusual. Fusion of spermatids to form giant cells was observed by 12 hours. CDB-4022 acts acutely on Sertoli cells to induce marked cellular rarefaction and degeneration, but not necrosis. A rapid and direct effect of CDB-4022 on spermatocytes and spermatids was observed. The antispermatogenic activity of CDB-4022 appears to be a consequence of direct effects on Sertoli and germ cells.

     Key words: Antifertility, morphology, ultrastructure, seminiferous epithelium, germ cells

	Materials and Methods

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Animals

Adult male Sprague-Dawley CD rats (Crl:CD[SD]; 250–275 g) were purchased from Charles River Laboratories (Kingston, NY). Rats were individually housed in polycarbonate solid-floor cages with Bed-o-Cob bedding (Andersons Industrial Products Group, Maumee, Ohio) and received laboratory rodent diet (Purina 5001) and tap water ad libitum. The photoperiod was a 14:10-hour light:dark cycle. The environmental conditions of the animal rooms were maintained as recommended (National Research Council, 1996) to the maximum extent possible. All study protocols were approved by BIOQUAL's institutional animal care and use committee.

Materials

CDB-4022 ([4aRS,5SR,9bRS]-2-ethyl-2,3,4,4a,5,9b-hexahydro-8-iodo-7-methyl-5-[4-carbomethoxyphenyl]-1H-indeno[1,2-c]pyridine-hydrochloride; also known as RTI-4587-073) was synthesized at Southwest Foundation for Biomedical Research (San Antonio, Tex) and was considered >99% pure based on high-performance liquid chromatography analysis. As in previous reports (Hild et al, 2001a,b, 2004), CDB-4022 was a racemic mixture of l and d enantiomers. DPP was purchased from Chem Service Inc (West Chester, Pa) and had a reported purity of 99%. Needles, syringes, anesthetic (ketamine and xylazine), and surgical supplies were purchased from NLS Animal Health (Baltimore, Md). Formaldehyde, glutaraldehyde, and other reagents or molecular biology–grade chemicals were purchased from Sigma-Aldrich (St Louis, Mo). Food-grade sesame oil (Hain) was purchased from a local grocery store.

Treatment of Animals and Tissue Processing

Adult male rats (4/time point) received a single oral dose of either vehicle (100% sesame oil, 5 mL/kg), CDB-4022 (12.5 mg/kg), or DPP (2200 mg/kg). DPP was included as a known Sertoli cell toxicant. Doses of CDB-4022 and DPP that induce maximal testicular damage as determined in previous studies (Creasy et al, 1983, 1987; Hild et al, 2001b) were used to determine acute testicular effects. The dose of CDB-4022 used in this study had previously been shown to induce maximal testicular weight loss and disruption of all seminiferous tubules in treated rats 7 days after a single oral dose was administered (Hild et al, 2001b). A twofold higher dose of CDB-4022, 25 mg/kg, resulted in no further reduction in testicular weight or morphology. At 3, 6, or 12 hours after oral dosing with CDB-4022 in this study, the rats were injected intraperitoneally with heparin (150 IU/kg) and anesthetized. Rats were then perfused through the heart as described by Sprando (1990) with McDowell and Trump 4% formaldehyde and 1% glutaraldehyde (4F:1G) in 0.1 M phosphate buffer (pH 7.2–7.4; Dykstra et al, 2002). In a similar manner, rats dosed with vehicle or DPP were perfused with the 4F:1G fixative at 3 or 12 hours, respectively. After perfusion, the fixed testes were removed, sliced as necessary, and placed in vials containing the 4F:1G fixative for 1 to 2 hours at room temperature. Vials were subsequently stored at 4°C and sent to the Laboratory for Advanced Electron and Light Optical Methods at North Carolina State University. A portion of the testis was embedded in glycol methacrylate (GMA) medium, and serial cross-sections (2 µm) were stained with Harris hematoxylin only or hematoxylin and Lee for evaluation at the light microscopic level.

Testicular tissues were further processed for ultrastructural evaluation by transmission electron microscopy as described previously by Dykstra et al (2002). Briefly, portions of the fixed testis were postfixed in 1% osmium tetroxide in 0.1 M phosphate buffer (pH 7.2–7.4), dehydrated in an ethanolic series culminating in 100% acetone, and infiltrated with Spurr epoxide resin. After polymerization overnight at 70°C, semithin sections (0.5 µm) were stained with 1% toluidine blue O in 1% sodium borate and examined with a light microscope. Areas of interest were selected and the blocks trimmed accordingly. Ultrathin sections (80–90 nm) were cut, placed on 200 mesh copper grids, and stained with uranyl acetate and lead citrate. Grids were viewed using a transmission electron microscope and the results documented photographically.

Testicular sections obtained from vehicle-treated rats were evaluated in conjunction with those from CDB-4022– and DPP-treated rats. These GMA or epoxide resin sections were used to establish normal testicular histology and potential fixation and processing artifacts at both the light and ultrastructural levels. The findings we describe in CDB-4022– and DPP-treated rats were different than those in the tissues obtained from the controls.

	Results

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Light Microscopic Observations

Normal morphology of the seminiferous tubules, including all stages of the spermatogenic cycle, was observed in the GMA (Figure 1A) and epoxide resin sections from vehicle-treated rats. As reported in a previous study (Hild et al, 2001a), vacuolization of Sertoli cells was observed in the basal compartments of seminiferous epithelium in both GMA- and epoxide resin–embedded testicular sections from CDB-4022–treated rats and was not observed in the testes of vehicle-treated rats. Vacuolization was observed as early as 3 hours postdosing with CDB-4022 (Figure 1B). At 6 and 12 hours (Figure 1C and D, respectively), there was an increase in both the extent of Sertoli cell vacuolization and the number of seminiferous tubules affected, a result that agrees with the previous report (Hild et al, 2001a). Vacuolization was apparent along the basement membrane and was associated with germ cells but was more prevalent around spermatocytes and spermatids. By 12 hours, vacuolization of Sertoli cell cytoplasm had increased to create clear areas adjacent to or within Sertoli cells and their nuclei appeared swollen as suggested by the lack of nuclear indentations and their rounded profiles compared with those observed in control animals. The chromatin in the nuclei of round spermatids contained clear spaces (Figure 1D). Detachment of germ cells from the seminiferous epithelium was observed in the GMA-embedded testicular sections from CDB-4022–treated rats. The testes of CDB-4022– and DPP-treated rats exhibited similar degenerative changes; however, the extent of damage in the DPP-treated rats at 12 hours was not as extensive as that observed 12 hours after CDB-4022 treatment. Although germ cell detachment was apparent in GMA sections, there was no clear separation of germ cells from Sertoli cells in the epoxide resin embedded testicular sections, suggesting this was an artifact in GMA processing of tissues. However, a centralized displacement of spermatocytes and spermatids, indicative of germ cell depopulation, was apparent in the epoxide resin embedded testicular sections from CDB-4022– and DPP-treated rats at 12 hours postdosing (not shown).

View larger version (156K): [in this window] [in a new window]   Figure 1. Glycol methacrylate–embedded testicular sections from a (A) vehicle-treated rat at 3 hours postdosing or (B) CDB-4022–treated rats at 3 hours, (C) 6 hours, or (D) 12 hours after dosing. Vacuolization (V) of Sertoli cell cytoplasm was apparent by 3 hours after dosing with CDB-4022 (B) and was more prominent at 6 hours (C) and 12 hours (D) after dosing. Clear spaces were also present in the nuclei of round spermatids (arrowheads), and the lumen of seminiferous tubules was occluded with germ cells by 12 hours. Bar = 50 µm.

View larger version (137K): [in this window] [in a new window]   Figure 2. Ultrastructural characteristics of testes from vehicle-treated rats. (A) Normal progression of germ cell development and Sertoli cell structure was observed. Magnification, 2600x. (B) Higher magnification of a basally located Sertoli cell. Magnification, 6500x. BM indicates basement membrane; M, mitochondria; Nu, nucleolus; L, lipid droplet; and SER, smooth endoplasmic reticulum.

View larger version (169K): [in this window] [in a new window]   Figure 3. (A) Three hours after CDB-4022 treatment Sertoli cells had intact nuclei, but a nucleolus was lacking and SER was dilated. Cytoplasmic rarefaction was so severe in these tubules that a clear space is apparent in the Sertoli cell just above the BM. Magnification, 2600x. (B) Three hours after CDB-4022 treatment swelling and disruption of the nuclear envelope, loss of electron density, and acrosome displacement (arrowhead) were observed in spermatids. Magnification, 2600x. (C) Three hours after CDB-4022 treatment nuclear swelling of spermatocytes resulted in disruption of the nuclear envelope such that 2 membranes were distinguishable (arrowheads and inset arrowhead). In addition, a thick layer of cytoplasm was observed between the outer acrosomal membrane and plasma membrane of spermatids (arrow andinset arrow). The spermatid plasma membrane was also not adjacent to the Sertoli cell membrane, and the putative acrosomes were not oriented toward the BM. Magnification, 2600x. Magnification for insets, 6500x. (D) TJ between Sertoli cells were disrupted, and V of Sertoli cell cytoplasm was prominent 6 hours after dosing with CDB-4022. Magnification, 4800x. BM indicates basement membrane; PS, pachytene spermatocyte; SER, smooth endoplasmic reticulum; TJ, tight junctions; and V, vacuolization.

In contrast, there were notable degenerative changes in Sertoli cell ultrastructure in CDB-4022–treated rats by 3 hours (Figure 3). In general, Sertoli cell nuclei remained intact, but there was an apparent decrease in the presence of nucleoli (Figure 3A). The most notable change in Sertoli cells at 3 hours was an increase in amount and diameter of swollen SER along with a reduction in cytoplasmic ground substance (Figure 3A and B). Cytoplasmic rarefaction, cellular disruption involving loss of cytoplasm and cytoplasmic membranes, was observed in the more severely affected Sertoli cells. This disruption and swelling resulted in clear areas adjacent to or within the Sertoli cells, particularly in the basal region of the cell (Figure 3A). Although A and B spermatogonia appeared unaffected by treatment with CDB-4022, spermatocytes and spermatids presented with abnormal morphology. In CDB-4022–treated rats, swelling and disruption of the nuclear envelope and a loss of electron density were observed in spermatocytes and spermatids within 3 hours after dosing (Figure 3B and C). In some spermatocytes, the swelling of the nuclear envelope was so severe that 2 membranes were easily distinguished (Figure 3C inset). Step 8 spermatids were especially noteworthy because chromatin was more diffuse and rarefied; the nuclear envelopes were incomplete, broken, or missing portions; and the position of the spermatid nucleus within the cell and relative to Sertoli cell cytoplasm was unusual. A thick layer of cytoplasm intervened between the outer acrosomic membrane and the plasma membrane, which are typically located in close proximity to each other. In turn, the spermatid plasma membrane was not adjacent to the Sertoli cell membrane as observed in the testes of vehicle-treated rats. The putative acrosomes in the Step 8 spermatids were also not oriented toward the basement membrane, as usually occurs at this developmental step.

The severity of the degenerative effects on Sertoli cells, as determined by degree of change and number of seminiferous tubules affected, increased at 6 and 12 hours after CDB-4022 treatment. By 6 hours, dilation of SER was more pronounced, more Sertoli cells demonstrated cytoplasmic rarefaction, and tight junctions between adjacent Sertoli cells were disorganized (Figure 3D). This disorganization was characterized by a lack of the typical hexagonal arrangement of actin filaments and a swelling of the associated ER. CDB-4022 effects on spermatocytes and spermatids at 6 hours (Figure 3D) were similar to those observed at 3 hours; however, by 12 hours (Figure 4), more severe disruption was evident. The chromatin of some spermatocytes was more electron dense and, in some nuclei, appeared to have clumped. Fusion of spermatids to form giant cells, cells with multiple nuclei, was also observed and appeared to be secondary to retraction of Sertoli cell processes (Figure 4A and B). Large open spaces in the cytoplasm of Sertoli cells were apparent by 12 hours (Figure 4B); these would appear as vacuoles at the light microscopic level (Figure 1D). Swollen mitochondria with electron lucent areas, an indication of early cellular degeneration, were observed (Figure 4C). In some Sertoli cells, the mitochondrial degeneration was characterized by swelling and enlargement of cristae, resulting in a "moth-eaten" appearance. In addition, membrane disorganization became more severe, accumulation of cellular debris in Sertoli cell cytoplasm was apparent, and phagocytic structures were observed.

View larger version (189K): [in this window] [in a new window]   Figure 4. Twelve hours after CDB-4022 treatment (A) fusion of germs cells due to loss of Sertoli cell processes was apparent and (B) spermatocytes fused to form multinucleated gc. Magnification, 2600x. After 12 hours of treatment Sertoli cell M exhibited signs of swelling and degeneration in both (C) CDB-4022– and (D) DPP-treated rats. Magnification, 6500x. Degeneration of spermatocytes and spermatids became more severe as nuclear envelopes were broken, and chromatin clumping was apparent (A and B). In addition, swollen SER, V, loss of cytoplasmic ground substance, and disrupted membranes and TJ were observed in Sertoli cells of DPP-treated rats (D). DPP indicates di-n-pentylphthalate; gc, giant cells; SER, smooth endoplasmic reticulum; TJ, tight junctions; M, mitochondria; and V, vacuolization.

	Discussion

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The indenopyridine CDB-4022 induced marked cellular rarefaction, plasma membrane disruption, disorganization of tight junctions, swelling of SER and mitochondria, and degeneration in Sertoli cells. The onset of these ultrastructural changes in Sertoli cells was rapid—they occurred within 3 hours after rats received a single oral dose of 12.5 mg/kg CDB-4022. In addition, degenerative changes in spermatocytes and spermatids were already observed at 3 hours after CDB-4022 treatment. These ultrastructural changes in germ cells were observed earlier than the detection of nuclear DNA fragmentation, which occurred 6 to 12 hours postdosing in a previous study (Hild et al, 2001a). The rapid effects on Sertoli cell ultrastructure are consistent with the notion that CDB-4022 is a Sertoli cell toxicant, and the results support our hypothesis that the Sertoli cell is a primary target of CDB-4022 action in the rat. Nonetheless, the early effects observed on germ cells suggest that CDB-4022 has direct effects on spermatocytes and spermatids. Since the effects on Sertoli and germ cells were observed concurrently, we are unable to determine the exact contribution of Sertoli cell disruption vs germ cell necrosis to the CDB-4022–induced depopulation of the seminiferous epithelium because the loss of Sertoli cell function also results in adverse effects on germ cells (Boekelheide et al, 2005). Similar ultrastructural changes in Sertoli and germ cells were previously observed in adult male rats after treatment with an indenopyridine analog, 20-438 (Hausler and Hodel, 1979). The dose of 20-438, a less potent indenopyridine, was 50 mg/kg, and testes were evaluated at 10, 24, or 48 hours postdosing. Sertoli cell changes included vacuolization of the ER and mitochondrial swelling by 10 hours after dosing. Nuclear malformations of spermatids were also observed as early as 10 hours postdosing with 20-438. These effects were more pronounced at 24 and 48 hours with disruption of Sertoli and germ cell contacts observed at 48 hours. These effects are consistent with our findings for CDB-4022.

The vacuolization of Sertoli cells observed at the light microscopic level is due to swelling and coalescence of intracellular membrane–bound organelles, particularly ER and vesicles (Chapin et al, 1983; Creasy et al, 1983, 1987). We observed similar distention of intracellular organelles, particularly the SER, in Sertoli cells after CDB-4022 treatment, which concurs with the vacuolization observed at the light microscopic level in this study. In addition to vacuolization, Sertoli cell membranes and tight junctions were disrupted. This decrease in membrane stability and disruption of tight junctions, a key component of the blood-testis barrier (Russell and Peterson, 1985), probably produced a fluid/ion imbalance (Creasy et al, 1987). CDB-4022 results in a dramatic decrease in STF secretion (Hild et al, 2001a), and loss of STF secretion may be due to perturbation of ion transporter activity (Johnson et al, 1991). The Sertoli cell toxicant DPP also caused structural and functional instability of the Sertoli cell plasma membranes, which has been proposed to lead to a loss of fluid/ion balance (Creasy et al, 1987). As seen in the present study, adverse effects on mitochondria were observed in Sertoli cells of both CDB-4022– and DPP-treated rats, which suggest metabolic injury. Since luminal germ cells require lactate produced by Sertoli cells and secreted in STF to prevent apoptosis and thereby promote germ cell survival (Jutte et al, 1981, 1982; Erkkila et al, 2002), disruption of SER function and STF secretion would affect the interaction between Sertoli and germ cells and result in germ cell death.

The structural changes in Sertoli cells of CDB-4022–treated rats indicate that CDB-4022 may also disrupt adhesion contacts between Sertoli and germ cells. The lonidamine analog AF-2364, an antispermatogenic compound also being investigated for use as a potential nonhormonal male contraceptive, has been shown to affect adhesion sites between Sertoli and germ cells (Lee et al, 2004). Within 4 hours after dosing with 50 mg/kg AF-2364, intercellular space was observed between germ cells, particularly round spermatids, and Sertoli cells at the electron microscopic level. By day 5, a noticeable increase in these spaces was observed. Similar ultrastructural findings between Sertoli and germ cells were observed within 12 hours of CDB-4022 treatment in the current study. Further investigation suggested that AF-2364 acts on the multiprotein complex of the ectoplasmic specialization, a testis-specific cell-cell actin-based adherens junction, with activation of pathways that end in depopulation of the seminiferous tubules (Cheng et al, 2005). To determine whether CDB-4022 activates similar pathways to cause depopulation of germ cells would require additional experiments.

In conclusion, the results of this study indicated that CDB-4022 acts rapidly on Sertoli cells to induce marked cellular rarefaction and degeneration but not cell death. The data also suggested a rapid and concurrent direct effect of CDB-4022 on spermatocytes and spermatids. However, some of the effects on germ cells observed in this study, particularly sloughing of germ cells, may still be due to indirect actions through Sertoli cells. Thus, the antispermatogenic activity of CDB-4022 appears to be a consequence of direct effects on both Sertoli and germ cells. Additional studies are needed to fully elucidate the mechanism of action of CDB-4022.

	Acknowledgments

 
The authors express thanks for the technical expertise of the following BIOQUAL technicians: Eileen Curreri, David Gropp, and Bruce Till. Care of the animals was provided by Abraham Elescano and Stephen Greenwell. We would also like to thank Vivian English of Experimental Pathology Laboratories Inc for processing of GMA sections and Dr Barbara Attardi for reviewing the manuscript. We thank Dr Richard Blye of the Contraception and Reproductive Health Branch, National Institute of Child Health and Human Development, for his input into the design of this study and review of the manuscript.

	Footnotes

 
This work was supported by National Institute of Child Health and Human Development contract NO1-HD-2-3338 awarded to BIOQUAL Inc.

This work was presented in part at the 38th Annual Meeting of the Society for the Study of Reproduction, July 24–27, 2005, Quebec City, Canada; Abstract 28.

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This Article Abstract Full Text (PDF) All Versions of this Article: 28/4/621    most recent Author Manuscript (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hild, S. A. Articles by Marshall, G. R. Search for Related Content PubMed PubMed Citation Articles by Hild, S. A. Articles by Marshall, G. R.