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Testis |
Division of Reproductive Biology, Department of Biochemistry and Molecular Biology, The Johns Hopkins University, Bloomberg School of Public Health, Baltimore, Maryland 21205
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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apoptosis, spermatogenesis, testosterone
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It is now established that lowering of intratesticular testosterone concentration results in the apoptotic death of some germ cells (e.g., pachytene spermatocytes) in association with nuclear DNA fragmentation in the dying cells [3, 9, 10]. However, other germ cells (e.g., round spermatids) undergo a loss of adhesion to the Sertoli cell, slough into the lumen of the seminiferous tubules [11, 12], and are phagocytized by Sertoli cells. These sloughed cells do not necessarily show the DNA fragmentation that is characteristic of classical apoptosis [13, 14], but rather appear to die because of the loss of attachment to the Sertoli cell.
Anoikis [15] is the term used for cellular death in response to the loss of attachment of an attachment-dependent cell from its basal lamina. The Bcl2 modifying factor (Bmf) is a BH3-only pro-apoptosis member of the Bcl2 family of apoptosis-related proteins [16]. It is the only known actin-associated apoptosis protein that initiates a cellular death cascade in response to a cell's loss of attachment from its basal lamina. As indicated above, some spermatids detach from the Sertoli cell in response to reductions in intratesticular testosterone concentration [11, 12]. In light of the role of Bmf in anoikis in other systems, it seemed likely to us that Bmf would be expressed in germ cells, and that its expression and normal distribution would be altered under conditions that induce widespread germ cell loss.
The results presented herein show that Bmf is, indeed, expressed in the normal rat testis. We show that it is highly conserved relative to the mouse and human homologues and is restricted in its distribution to the subacrosomal space of postmeiotic germ cells between steps 4 and 16 of spermiogenesis. In response to lowered intratesticular testosterone levels, Bmf mRNA expression and protein distribution were found to change dramatically. Specifically, the steady-state mRNA levels increased markedly in germ cells, the protein redistributed throughout the germ cells (rather than being restricted to the subacrosomal space of spermatids), and germ cells in which Bmf was not normally expressed were found to express the Bmf protein. We suggest that Bmf is likely to play an important role in germ cell death in response to reduced intratesticular testosterone.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Male Sprague-Dawley rats of 8–12 weeks of age were purchased from Charles River (Kingston, MA). All rats were housed in a vivarium under a 14L:10D cycle and provided water and rat chow ad libitum. To experimentally suppress LH-stimulated testosterone production from Leydig cells, rats were administered subdermal 2.5-cm testosterone (T) and 0.1-cm 17ß-estradiol (E)-filled polydimethylsiloxane (Silastic; Dow Corning, Midland, MD) capsules for 14 or 56 days according to methods previously described [17]. Control rats received empty capsules. This protocol was approved by the Johns Hopkins University Animal Care and Use Committee.
Testicular Isolation and Cloning of the Rat Bmf cDNA
RNA was purified from adult rat tissues by the Trizol method (Invitrogen Corporation, Carlsbad, CA). A testicular cDNA library was generated by performing first-strand synthesis from total RNA. Briefly, RNA (3 µg) was reverse transcribed in a 20 µl reaction at 46°C for 60 min using 0.2 units of Superscript II (Invitrogen) and 50 ng of oligo-dT primer in single-strength, first-strand synthesis buffer according to manufacturer's specifications.
The rat Bmf cDNA was cloned via a degenerate polymerase chain reaction (PCR)-based strategy. Primers were designed corresponding to the conserved 5' Bmf dynein light chain-binding motif [5'-CAC TCA GAC CCT CAG TCC A-3' (sense)] and the 3' BH3 region [5'-GCC GAT GGA ACT GGT CTG CAA-3' (antisense)] of the mouse cDNA sequence (GenBank accession NM_138313) so as to amplify an approximately 230-base pair (bp) fragment of the rat Bmf cDNA. PCR was performed in a reaction volume of 50 µl containing 0.5 µl of the reverse transcription (RT) reaction, single strength buffer, 20 µM dNTPs, 1.5 mM MgCl2, 400 nM forward primer, 400 nM reverse primer, and 0.5 units AmpliTaqR DNA Polymerase (Perkin Elmer, Boston, MA). The PCR conditions were 35 cycles at 94°C for 30 sec, 58°C for 30 sec, and 72°C for 1 min, and a final extension of 72°C for 2 min. The 5' and 3' rapid amplification of cDNA ends (RACE) reactions were then performed using the 5' RACE System for Rapid Amplification of cDNA Ends Reagent Assembly kit, Version 2.0 (Invitrogen) according to manufacturer's specifications. PCR products were cloned into a p-GemT Easy Vector (Promega, Madison, WI) according to manufacturer's specifications and sequenced to verify insert product. A BLAST search was performed on the sequences to verify homology to human and mouse Bmf. During the cloning of the rat cDNA, the full-length rat Bmf mRNA became available (GenBank accession AF506761) through NCBI. The cDNA and predicted protein sequences found in the present study were 100% identical to this sequence.
Immunofluorescence Microscopy
For immunofluorescence studies of testis sections, rats were anesthetized and whole-body perfused with neutral buffered formalin for 1 h at a rate of 7 ml/min. The testes were removed and immersed in neutral buffered formalin overnight at 4°C. The tissue was then dehydrated in ice cold (4°C) 70%, 90%, and 99% ethanol for 1 h each, and then in absolute ethanol for 1 h at room temperature. Tissue was infiltrated with 50% polyester wax/50% ethanol for 2 h at 42°C followed by a 90% polyester wax/10% ethanol mixture for 1 h at 42°C. The tissue was then transferred into 90% wax/10% ethanol in plastic embedding dishes and chilled on ice for 30 min or until the wax solidified. Sections (5 µm) were cut and mounted on Hipure (Norland Products, Cranbury, NJ) subbed glass slides. The slides were dewaxed by immersion into 100%, 90%, and 70% ethanol baths for 10 min each. Slides were blocked in PBS-diluted normal horse serum (1:60, Vector Laboratories Inc., Burlingame, CA) and then incubated (1 h, room temperature) with a polyclonal antibody raised against the N-terminus of the human Bmf protein in rabbit (1:200; Abcam Ltd., Cambridge, UK). The N-terminal sequence of human Bmf used for antibody production differs from that of the N-terminal sequence of the rat by one amino acid. Bound primary antibodies were detected with a fluorescein 5(6)-isothiocyanate (FITC)-conjugated anti-rabbit immunoglobulin (Ig)M secondary antibody raised in goat (1:100; Vector) or a Texas Red-conjugated anti-rabbit IgM secondary antibody raised in goat (1:100; Vector). Nuclei were stained with Vectashield Anti-Fade Mounting Medium containing 4',6-diamidino-2-phenylindole (DAPI) (Vector). Primary antibody specificity was demonstrated by performing the above immunostaining procedure preceded by the (preabsorption) reabsorption of equal concentrations of the Bmf primary antibody with the Bmf N-terminal peptide used for antibody production (EPSQCVEELEDDV, amino acids 2–14 of human Bmf; Abcam) for 30 min at 37°C. In contrast to tissue sections incubated with the primary antibody, tissue sections incubated with the preabsorbed Bmf antibody yielded no detectable immunofluorescence. Acrosome staining was performed using the acrosome-specific lectin, peanut agglutinin (PNA), conjugated to FITC (Sigma, St. Louis, MO). Images were obtained by a Nikon Microflex H-III automatic camera system (Nikon Corporation, Tokyo, Japan), with a 20x or 40x Zeiss PlanApo lens.
Electron Microscopy
Testes were fixed by whole-body vascular perfusion with 2.5% glutaraldehyde in 0.1 M cacodylate buffer. Tissue cubes were postfixed in cacodylate-buffered 1% osmium tetroxide and embedded in Epon. For electron microscopic analysis, 600–800 nm sections were cut with a diamond knife. Thin sections were mounted on 200-mesh grids and stained with uranyl acetate and lead citrate.
Germ Cell Isolation and Separation by STAPUT
Total germ cells were isolated from untreated and 14-day testosterone (T)- and estradiol (E)-treated animals based on the procedure used by Goldberg et al. [18]. Briefly, testes were removed and decapsulated, then placed in cold (4°C) Krebs buffer (125 mM NaCl, 3 mM KCl, 1 mm NaH2PO4, 1.2 mM MgSO4, 2.4 mM CaCl2, 22 mM NaHCO3, and 10 mM glucose). Collagenase (25 mg; Type 1A; Sigma) was added and incubated in a 33°C shaking water bath for 20 min or until the tubules became dissociated. The tubules were washed three times with fresh, cold KREBS. DNAse I (100 µg/ml; Sigma) and trypsin (25 mg; Sigma) were added and incubated in a 33°C shaking water bath for 20 min. Tubules were disrupted mechanically by pipetting followed by filtering through 80 µm mesh. Cells were washed with PBS and then spun at 1500 rpm (three times). The isolated germ cells were then counted on a hemacytometer.
The cells were separated by loading 8.8 x 108 total germ cells into a 12.5-cm-diameter STAPUT chamber (GlassShop, ProScience, Scarborough, ON, Canada). Separation took place on a 1-L linear BSA (Sigma) gradient (2%–4%) for 2 h. Fractions (15 ml) were collected over 1 h and examined under light microscopy to identify those enriched in pachytene spermatocytes and round spermatids. These fractions were then pooled. The purity of STAPUT-isolated germ cell fractions was assessed by microscopic analysis and by Northern blot analysis (see below).
Northern Blot Analysis
Purity of STAPUT-isolated germ cell fractions was assessed by Northern blot analysis. RNA was purified from isolated pachytene spermatocytes and round spermatids by the Trizol method (Invitrogen) as above. Total RNA (10 µg) from each germ cell preparation was fractionated in a 1% agarose/formaldehyde gel; transferred overnight to a nylon membrane (HybondTM-N, Amersham Pharmacia, Piscataway, NJ); and UV cross-linked (UV Stratagene 1800). Complementary DNA fragments of hemiferrin and protamine 2 [19] were radiolabeled with (
-32P) dATP using the Rad Prime DNA Labeling Kit (Invitrogen). These genes were chosen to determine germ cell purity in STAPUT-isolated fractions because hemiferrin expression is seen in both pachytene spermatocytes and round spermatids [20], whereas protamine 2 mRNA expression is seen only in round spermatids [21]. Northern blots were hybridized overnight at 65°C with labeled cDNA probes in ExpressHyb hybridization solution (Clontech, Palo Alto, CA). Following hybridization, blots were washed in 2x sodium chloride-sodium citrate (SSC)/1.0% SDS for 30 min at 65°C; 1x SSC/0.5% SDS for 30 min at 65°C; and 0.1% SSC/0.1% SDS for 30 min at 65°C. After blots were washed, they were placed in a phosphor screen cassette for 8–12 h. The signals were detected using a Typhoon 8600 and ImageQuant software (Amersham).
Semi-Quantitative RT-PCR of Bmf mRNA
RNA was purified from isolated pachytene spermatocytes, round spermatids, and elongated spermatids by the Trizol method (Invitrogen), and cDNA synthesis was performed as above. PCR reactions utilized 1.5 µg of cDNA, and the reaction conditions for both Bmf and the loading control, ribosomal protein L19, were performed as above. Gene-specific primers for ribosomal protein L19 were 5'-GGACAGAGTCTTGATGATCTC-3' (sense) and 5'-CTGAAGGTCAAAGGGAATGTG-3' (antisense) to amplify a 195-bp fragment corresponding to the region 401–595 of the L19 cDNA (GenBank accession NM_031103). PCR products (10 µl) were separated on a 1.5% agarose gel and visualized with ethidium bromide.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A degenerate RT-PCR-based strategy was adopted to clone the rat homologue of the Bmf cDNA and to determine if Bmf is expressed in the rat testis. A 235-bp PCR product was generated from rat testicular, ovarian, kidney, and liver cDNA libraries using primers designed to correspond to the conserved mouse Bmf 5' dynein light chain binding domain and the 3' BH3 domain. The sequence obtained was subjected to a BLAST search, which confirmed that it corresponded to the mouse and human homologues of Bmf. The 5' and 3' portions of the rat Bmf cDNA were obtained by RACE (Fig. 1). As seen in Figure 1, the mouse, human, and rat homologues of Bmf are highly conserved, with the predicted protein sequence of the rat being 96% and 87% identical to the protein sequences of the mouse and human, respectively. The dynein light chain and BH3 domains were 100% conserved among the three homologous proteins.
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Immunolocalization of Bmf in the Rat Seminiferous Epithelium
Figure 2 depicts Bmf localization (green) in sections of rat testis at particular stages (VI, II–III, IV, V, VII, XI, and XIII) of the cycle of the seminiferous epithelium. The stages of spermatogenesis were determined by viewing these sections by phase contrast microscopy (not shown). At stage VI, Bmf expression was restricted to postmeiotic cells, specifically step 6 round spermatids. Staining was absent from the meiotic pachytene spermatocytes and from the step 18 elongated spermatids at this stage. Staining for the Bmf protein in the step 6 spermatids was polarized, reminiscent of the region and shape of the developing acrosome. Bmf expression was absent from step 2–3 round spermatids at stages II–III, but appeared faintly at the very tips of the step 16 elongated spermatids. At stage IV, Bmf staining was localized to the area of the acrosomal granule of step 4 round spermatids and absent from step 17 elongated spermatids. At stages V and VIII, Bmf staining appeared to be associated with the developing acrosome of the step 5 and 8 spermatids, respectively, and at both stages staining was absent from the elongated spermatids (steps 17 and 19, respectively). At stage XI, Bmf localized around the elongating nuclei of the step 11 spermatids. Finally, at stage XIII, Bmf was seen predominantly at the tips of the step 13 elongated spermatids, and also around the periphery of the nuclei of a few spermatids. Taken together, these observations indicate that testicular Bmf protein expression is restricted to the region of the developing acrosome of postmeiotic spermatids between steps 4 and 16 of spermiogenesis.
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Given the polarized distribution of the Bmf protein in spermatids, in what appears to be the region of the developing acrosome, we more closely examined Bmf localization in relationship to the acrosome. Figure 3 is a stage VII tubule double labeled with the acrosome-specific lectin, peanut agglutinin (green), and with Bmf (red). The arrows point to distinct areas in which it is apparent that staining for the acrosome and for Bmf are adjacent, not overlapping. This can be seen clearly in the higher magnification figure inset, which shows a distinct region of Bmf protein distributed subadjacent to the acrosome. The region of Bmf staining is known as the subacrosomal space [22] and is located directly between the germ cell nucleus and the acrosome.
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Distribution of Bmf Under Conditions of Experimentally Reduced Intratesticular Testosterone Concentrations
With the knowledge that Bmf is present in testicular germ cells and that it is involved in anoikis in other systems [16], we tested the hypothesis that Bmf might undergo changes in expression, distribution, or both under conditions in which germ cells were induced experimentally to undergo cell death. To test this hypothesis, adult rats were administered testosterone and estradiol (TE)-containing Silastic capsules designed to induce germ cell apoptosis or anoikis by suppressing Leydig cell testosterone production [17]. As shown in Figure 4, Bmf localization changed dramatically by 8 weeks of TE treatment. Thus, whereas Bmf normally localized to the subacrosomal area of round spermatids (see Fig. 2), the protein was redistributed throughout germ cells in response to reduced intratesticular testosterone. Figure 4A shows Bmf localization in a typical seminiferous tubule from rats that had received TE implants for 8 weeks, and Figure 4B shows the same tubule with Bmf (red) and DNA (blue) merged. The Bmf-stained nuclei were situated close to the tubule lumen, where pachytene spermatocytes reside following the extensive germ cell loss brought about by the TE treatment. Figures 4C–E show the same tubule stained for Bmf (Fig. 4C), Bmf plus nuclei merged (Fig. 4D), and nuclei only (Fig. 4E), at higher magnification than the tubule shown in Figures 4A and 4B. These figures provide further evidence that Bmf staining occurs throughout the germ cells and is not polarized as it is in cells from control testis; and, moreover, that Bmf staining also occurs in spermatocytes, rather than being limited to postmeiotic spermatids. By comparing Figure 4F (phase contrast micrograph) with Figures 4C–E, it is evident that the germ cells that stain for Bmf exhibit a dark and rounded-up morphology compared with cells that are unstained, suggesting that the Bmf-stained cells are undergoing programmed cell death.
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Although most germ cells fail to progress past the pachytene spermatocyte/early round spermatid stage following prolonged reduction of intratesticular testosterone, a few seminiferous tubules contain germ cells that have progressed to later steps of development. Examination of such tubules provided the opportunity to further scrutinize the effect of reduced testosterone on Bmf organization. Figures 5A and 5B show a tubule stained for Bmf (red) and for Bmf plus DNA (blue), respectively, in which some germ cells have progressed to step 7–8 round spermatids despite the reduced testosterone. As observed in the tubules from TE-treated animals that lack spermatids (see Fig. 4), Bmf protein expression was evident throughout some of the germ cells, in contrast to its polarized localization in the cells of untreated animals. The spermatid subacrosomal localization that characterizes control testes can also be seen in this tubule. Additionally, Bmf seems to be progressing outward from the subacrosomal space in some spermatids, suggesting a loss of subacrosomal localization. Figure 5 supports the contention that there is a change in the subacrosomal region of spermatids following reduced testosterone. Electron microscopy was utilized to examine the morphology of the subacrosomal space in germ cells from untreated animals and from animals subjected to 30 days of lowered intratesticular testosterone concentrations. Figure 5C is a representative electron micrograph of a Sertoli cell-germ cell junctional complex. A step 7 germ cell nucleus, the densely stained acrosome, and the subacrosomal space where Bmf is seen to localize are depicted. Also shown is the actin-based Sertoli cell junctional complex known as the ectoplasmic specialization, which consists of a cistern of smooth endoplasmic reticulum, hexagonally arrayed actin bundles, and the Sertoli cell plasma membrane [23]. Figure 5D depicts a Sertoli cell-germ cell junction following 1 month of reduced intratesticular testosterone. In many germ cells, the acrosomal granule failed to fuse with the developing acrosomal system as it normally does at step 4 of spermiogenesis [24], and the unfused granule was seen subadjacent to the developing acrosome. The effect of this was to cause the germ cell nuclear envelope to become invaginated, and thus create an enlargement of the subacrosomal space where Bmf normally resides. The germ cell also apparently lost adhesion to the Sertoli cell, as evidenced by the large gap between the two cells and the absence of the ectoplasmic specialization junction from the Sertoli cell. Thus, prolonged reduction of intratesticular testosterone can lead to an enlargement of the subacrosomal space where Bmf localizes under normal conditions.
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In summary, in response to the prolonged reduction of intratesticular testosterone levels, Bmf is expressed in cells at earlier developmental stages than the spermatid-only expression seen in untreated animals. This is accompanied by a change in Bmf localization from a predominantly subacrosomal position to a distribution throughout the cytoplasm and nucleus.
Bmf mRNA Expression in Specific Populations of Germ Cells from TE-Treated and Untreated Rats
The altered distribution of Bmf in germ cells following reduced testosterone concentration, and its appearance earlier in germ cell development, suggested that Bmf expression might change in response to testosterone changes. To test this, we examined Bmf mRNA levels in germ cells (pachytene spermatocytes and round spermatids) isolated by the STAPUT method from the testes of control rats and rats administered TE-containing capsules for 14 days. Northern blot analysis revealed that hemiferrin was present in both the isolated pachytene spermatocytes and round spermatid populations, but that protamine 2 was present only in the round spermatids (Fig. 6A). These results support the contention, based on morphological evaluation of the isolated germ cells, that the populations were isolated at high levels of purity. Semiquantitative RT-PCR was performed on RNA isolated from TE-treated and control cell populations to assess Bmf message levels under normal and conditions of reduced intratesticular testosterone. The results, shown in Figure 6B, were that Bmf message levels were very low in the pachytene spermatocytes and round spermatids isolated from control rats, but that Bmf mRNA was expressed at higher levels in both cell types with reduced levels of intratesticular testosterone. These results suggest that lowering of intratesticular testosterone results in an increase in Bmf expression in pachytene spermatocytes and round spermatids relative to levels in untreated controls in which basal expression is barely detectable.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the testes of untreated rats, Bmf was found to reside in the subacrosomal space of postmeiotic spermatids from steps 4 to 16 of spermiogenesis. Russell et al. [25] used immunoelectron microscopy to demonstrate that the subacrosomal space of developing spermatids is highly enriched in F-actin. The localization of Bmf to this region of the developing germ cell is therefore not surprising because, at steady state, Bmf is normally sequestered to the actin cytoskeleton via its conserved dynein light chain binding domain [16]. The actin polymers in the subacrosomal space have been shown to disappear at late steps of spermiogenesis, specifically step 19, just before release of the mature spermatozoa from the Sertoli cell and into the lumen of the seminiferous tubule [25]. Consistent with this, we have observed the absence of the Bmf protein from the subacrosomal space near the end of spermiogenesis, although at a developmental step (step 16) somewhat earlier than step 19. Thus it appears that Bmf may be degraded just before spermatids are released from the seminiferous epithelium as immunostaining is absent from the most mature spermatids, steps 15–19, and from mature spermatozoa. Given the role that Bmf plays in other systems as an initiator of cell death following loss of attachment of a cell from its basal lamina [16], the presence of Bmf in spermatids potentially ensures that any immature spermatid that inadvertently loses attachment to its underlying Sertoli cell will undergo Bmf-mediated apoptosis.
To determine the effect of reduced intratesticular testosterone on Bmf expression and distribution, intratesticular testosterone concentration was reduced by administering 2.5 cm testosterone- and 0.1 cm estradiol-containing Silastic capsules to the rats. Spermatogenesis can be maintained or restored quantitatively with 24 cm testosterone-/0.1 cm estradiol-containing capsules [6]. Therefore, we were confident that any effect of the 2.5 cm testosterone/0.1 cm estradiol capsules on Bmf would result from reduced testosterone and not from the sustained release of estrogen [6]. In fact, the expression and distribution of Bmf changed dramatically in the germ cells when intratesticular testosterone was reduced. In particular, there was earlier expression of the Bmf protein in meiotic spermatocytes, rather than only in postmeiotic spermatids. Moreover, there was a striking change in the localization of Bmf. Thus, whereas Bmf was seen only in the subacrosomal space of spermatids in untreated rats, this protein redistributed to become localized to both the nucleus and cytoplasm of germ cells when intratesticular testosterone was reduced. At the same time, the expression of Bmf mRNA increased in both pachytene spermatocytes and round spermatids under conditions of reduced intratesticular testosterone. This result is consistent with a recent study showing that, during differentiation, apoptosis-prone oligodendroglial lineage cells substantially increase their Bmf mRNA expression [26]. However, an earlier study reported that Bmf message levels were unaffected in cell cultures when Bmf-mediated apoptosis (anoikis) is induced in vitro [16].
Previous studies demonstrated that when a cell's actin cytoskeleton is perturbed as a consequence of loss of adhesion of the cell from its basal lamina, Bmf is released from the actin/myosin complex and is translocated to the mitochondria. There, Bmf interacts with the prosurvival Bcl2 protein via its BH3 domain [16], which interferes with the prosurvival function of Bcl2 and leads to the expulsion of cytochrome c from the inner mitochondrial membrane and into the cytoplasm. This, in turn, activates a caspase cascade, ultimately leading to the death of the cell. It is possible, therefore, that the altered Bmf expression pattern and distribution that occur in germ cells after the experimental reduction in intratesticular testosterone concentration may play a role in the initiation of an apoptotic cascade in germ cells by binding to and deactivating anti-apoptosis Bcl2 family members [16], by interacting with and activating pro-apoptotic Bcl2 family members [27, 28], or by affecting other members of the Bcl2 family [29, 30]. These possibilities have yet to be addressed, however, as has the potential relationship of Bmf to other possible mediators of germ cell death, such as the Fas-Fas ligand system [31–35].
Further complicating an understanding of the response of germ cells to reduced intratesticular testosterone concentration is that less mature cells, including meiotic spermatocytes and early round spermatids, exhibit nuclear DNA fragmentation as a hallmark of classical apoptosis, whereas more mature spermatids appear to undergo anoikis. However, the loss of spermatid adhesion due to reductions in intratesticular testosterone itself differs from the established definition of anoikis [15] in that, rather than a loss of cell-basal lamina adhesion occurring to bring about cellular death, the loss of adhesion occurs between two cell types: the germ cell and the Sertoli cell. How, or if, Bmf is involved with either or both processes remains to be determined.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 410 614 2356; mshow{at}jhsph.edu
Received: 11 September 2003.
First decision: 1 October 2003.
Accepted: 2 December 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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