DEVELOPMENTAL DYNAMICS 227:458 – 467, 2003
PATTERNS
Chronological Gene Expression of ADAMs During Testicular Development: Prespermatogonia (Gonocytes) Express Fertilin  (ADAM2) Carolina Rosselot, Abraham L. Kierszenbaum, Eugene Rivkin, and Laura L. Tres*
Immediately after birth, primordial germinal cell-derived prespermatogonia (PSG), located in the center of the testicular cords, migrate between adjacent Sertoli cells to establish contact with the cord basal lamina. PSG migration suggests continued assembly and disassembly of cell– cell contacts by a molecular mechanism that may involve integrins and their ligands, the disintegrin domain of spermatogenic cell-specific plasma membrane proteins called ADAMs. We have analyzed the temporal gene expression of selected ADAMs in intact fetal, early postnatal, and pubertal rat testis and Sertoli–spermatogenic cell cocultures by reverse transcriptase-polymerase chain reaction, in situ hybridization, and immunocytochemistry. We report that several ADAM transcripts are expressed in fetal, neonatal, and prepubertal testes. Cyritestin (ADAM3), ADAM5, ADAM6, and ADAM15 are expressed in day 17 fetal testes. In contrast, no expression of fertilin ␣ (ADAM1) and fertilin  (ADAM 2) was detected in fetal testes. Fertilin  gene expression starts after postnatal day 2, subsequent to the expression of fertilin ␣, which occurs on postnatal day 1. After postnatal day 2, all the indicated ADAMs, including the fertilin ␣ and fertilin , continue to be expressed. Transcripts of spermatogenic cell-specific fertilin ␣, fertilin , ADAM3, and ADAM5 were detected during the coculture of PSG with Sertoli cells for up to 72 hr after plating. The presence of fertilin  mRNA and protein in cocultured PSG was visualized by in situ hybridization and immunocytochemistry, respectively. These observations indicate that PSG in coculture with Sertoli cells provide a suitable approach for analyzing cell– cell adhesive responses involving spermatogenic cell-specific ADAMs. Developmental Dynamics 227:458 – 467, 2003. © 2003 Wiley-Liss, Inc. Key words: cell adhesion; cell migration; spermatogonia; Sertoli cell; spermatogenesis in vitro Received 21 February 2003; Accepted 21 April 2003
INTRODUCTION Soon after birth, mitotically arrested prespermatogonia (PSG; also called gonocytes) migrate from the center of the testicular cords to the periphery where they contact the basal lamina and resume mitotic activity (Peters, 1970; McGuinness and Orth, 1992). PSG migration may require cytoskeletal motive forces and a transient lessening of cell– cell adhe-
sion. Yet the precise mechanism remains largely unknown. We have focused our attention on a family of proteins, which combine features of both cell surface adhesive molecules and proteases as potential candidates involved in the translocation of PSG. ADAM (for A Disintegrin And Metalloprotease) belongs to a family of evolutionary conserved membrane proteins related
to snake venom integrin ligands and metalloproteases (reviewed by Blobel and White, 1992; Schlo ¨ ndorff and Blobel, 1999; Primakoff and Myles, 2000; Evans, 2001). ADAM proteins consist of several regions: a prodomain, and metalloprotease, disintegrin, cysteine-rich (usually containing an epidermal growth factor [EGF] repeat), transmembrane, and cytoplasmic domains.
Department of Cell Biology and Anatomical Sciences, The Sophie Davis School of Biomedical Education/The City University of New York Medical School, New York, New York Grant sponsor: National Institutes of Health; Grant number: HD36477. *Correspondence to: Dr. Laura L. Tres, Department of Cell Biology and Anatomical Sciences, The City University of New York Medical School, Harris Hall Suite 306, New York, NY 10031. E-mail:
[email protected] DOI 10.1002/dvdy.10327
© 2003 Wiley-Liss, Inc.
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Upon removal of the prodomain and the metalloprotease domain by endoproteolytic processing, the disintegrin domain is exposed at the N-terminal region and becomes available for cell– cell adhesion by binding to integrin ligands. The designation disintegrin refers to its property of disrupting integrin binding to classic ligands, including fibronectin and laminin. The ␣ and  subunits of the heterodimeric sperm protein fertilin (also designated ADAM-1 and ADAM-2, respectively; see http:// www.people.Virginia.edu/⬃jw7g/ Table_of_the_ADAMs.html for a table of ADAM gene and protein nomenclature) were the first members of the ADAM family to be recognized as spermatogenic cell-specific and implicated in sperm– egg membrane fusion (Primakoff et al., 1987). In addition to fertilin ␣ and fertilin , the gene expression of several members of the ADAM family has been detected in somatic and spermatogenic cell components of prepubertal and adult rat testis and other organs (Frayne et al., 1997). An ␣61 integrin receptor on mouse egg plasma membrane acts as the sperm receptor to which the disintegrin domain of fertilin  binds (Almeida et al., 1995). It has been reported that fertilin  but not fertilin ␣ displays adhesive activity by interacting with 1 integrin (Evans et al., 1997). Fertilin  and ␣ are synthesized as large precursors by spermatogenic cells (Lum and Blobel, 1997). The fertilin ␣ precursor is processed in the testis into the mature form by a proteolytic process. The fertilin  precursor is separately processed in the epididymis during sperm maturation. The fertilin ␣ and  heterodimer is detected on the posterior head region of sperm (Cowan and Myles, 1993) and is expressed in rat, rabbit, mouse, macaque, and guinea pig (Wolfsberg et al., 1995). An earlier in situ hybridization study of guinea pig and mouse testis has determined that the expression of fertilin ␣ and  transcripts begins during meiotic prophase, in pachytene spermatocytes. A more sensitive polymerase chain reaction (PCR) analysis of the developmental expression of the fertilin ␣ / complex during rat spermat-
ogenesis has revealed that fertilin ␣ is in fact detected from postnatal day 1 on, whereas fertilin  expression is visualized from postnatal day 19 on (Frayne et al., 1997). This reported chronology suggests that the full assembly of the fertilin ␣/ heterodimer occurs after postnatal day 19. Discrepancies still exist on the developmental expression of members of the ADAM protein family during testicular organogenesis and postnatal development. In fact, the potential role of ADAMs in testicular cell adhesion awaits elucidation. The latter is a relevant issue because the mitotic clonal expansion of the spermatogonial stem cell progeny occurs immediately after migration of PSG toward the basal lamina. Therefore, PSG and derived spermatogonial cell cohorts represent excellent model systems for defining the role of integrins and ADAMs during their interaction with the somatic Sertoli cells. Here we report the time course of expression of several ADAMs, including fertilin ␣ and . This study involved reverse transcriptase-PCR (RT-PCR), in situ hybridization, immunocytochemistry, and a cell coculture technique in which PSG interact with Sertoli cells. Three relevant observations derived from these studies. First, the testicular expression of fertilin ␣ and  mRNAs occurs soon after birth. Second, spermatogenic cell-specific cyritestin/ADAM3 and ADAM5 can be detected in fetal testis, thus preceding the expression of fertilin ␣ and . Third, fertilin ␣, fertilin , cyritestin/ADAM3, and ADAM5 transcripts are expressed in PSG–Sertoli cell cocultures.
RESULTS Temporal Expression of Selected ADAMs During Testicular Development Our first approach was to determine by RT-PCR whether fertilin ␣, fertilin , cyritestin/ ADAM3, ADAM5, ADAM6, and ADAM15 transcripts were expressed in fetal rat testis (when testicular cords consist of Sertoli cells and PSG are located in the core of the cords). The objective was to
compare the time course progression of ADAM gene expression in fetal testis with that observed soon after birth (postnatal days 1 and 2, when PSG initiate their translocation between adjacent Sertoli cells), and in the adult testis (when spermatogenesis is already established). Figure 1 indicates that only cyritestin/ ADAM3, ADAM5, ADAM6, and ADAM15 but not fertilin ␣ and fertilin  were expressed in the 17-day-old fetal testis. A significant, yet expected addition to this pattern was the expression of fertilin ␣ in the postnatal day 1 testis (Fig. 1). On postnatal day 2, fertilin  mRNA expression was unexpectedly added to the fertilin ␣, cyritestin/ADAM3, ADAM5, ADAM6, and ADAM15 complement. The expression of all the indicated ADAMs in the postnatal day 2 testis persisted in the adult testis (Fig. 1). Whole testes contain, in addition to testicular cords and seminiferous tubules, abundant blood and lymphatic vessels and connective tissue, potential sources of ADAM gene products. We reasoned that the cell-specific expression of ADAMs could be better determined by using a coculture system in which Sertoli cells and PSG are present. Therefore, our next approach was to establish first, whether selected ADAMs are expressed in cocultures prepared from 9-day-old rats (where Sertoli cell and spermatogonial cell progenies are present) and from 18day-old rats (where Sertoli cells coexisted with spermatogonial and spermatocytes progenies), and second, verify their time course expression, which could lead to future functional cell– cell adhesion studies. Figure 2 shows that the expression of fertilin ␣, fertilin , cyritestin/ADAM3, ADAM5, ADAM6, and ADAM15 transcripts occurs in the cocultures prepared from 9-day-old rat testes and detected for up to 72 hr. The expression of ADAMs beyond the 72-hr time period was not determined. A comparison of ADAM gene expression patterns with those observed in cocultures prepared from testes of 18-day-old rats yielded similar results (Fig. 3). Collectively, these studies indicated that (1) a brief asynchronous expression of fertilin ␣ and  mRNAs occurred in the early postna-
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Fig. 1. Developmental expression of selected ADAMs during rat testicular development (fetal day 17, postnatal days 1 and 2, and adult testis) detected by reverse transcriptase-polymerase chain reaction. Cyritestin/ADAM3, ADAM5, ADAM6, and ADAM15 genes are expressed during fetal day 17. The expression of fertilin ␣ and fertilin  genes was not seen. On postnatal day 1, all the disintegrins detected during fetal day 17 were visualized in addition to fertilin ␣. The expression of fertilin  was not observed. On postnatal day 2, fertilin  transcripts complemented ADAMs expressed on postnatal day 1. All ADAMs observed on postnatal day 2 continue to be expressed in the adult testis. Actin mRNA was used as positive control. Marker and amplicon values are indicated in kilobases (kb) and base pairs (bp), respectively. ADAM, a disintegrin and metalloprotease.
tal testis, (2) the expression of both fertilin ␣ and  takes place before the initiation of spermatogenesis, and (3) both fertilin ␣ and  mRNAs are expressed for at least three days in Sertoli–PSG and Sertoli–spermatogenic cell cocultures.
Temporal Expression of Fertilin  During Testicular Development and in Cocultures RT-PCR analyses pointed to the unanticipated early expression of fertilin  and other spermatogenic cellspecific ADAMs during testicular development and in Sertoli–PSG cocultures. An in situ hybridization approach was used to visualize first in whole testes the time course of expression of fertilin  and, second, to
determine which cells in the cocultures express fertilin . Figure 4A shows that a fertilin  antisense probe yields a positive reaction in whole testes collected from 1- to 7-day-old rats. Control (no oligonucleotide probe) and fertilin  sense oligonucleotide probe supported the specificity of the reaction produced by the antisense oligonucleotide. Figure 4B illustrates a Sertoli– PSG coculture prepared from 4-dayold rats in which the omission of the oligonucleotide probe failed to generate a color product. In contrast, Figure 4C demonstrates that fertilin  antisense oligonucleotide produces a positive reaction in selected PSG identified by phase contrast microscopic criteria (see below). Sertoli cells, which do not show a positive reaction, serve as a valuable inter-
nal negative control. We concluded from these observations that a close correlation existed between the RTPCR and in situ hybridization data concerning the early postnatal testicular expression of fertilin  mRNA and that the cocultured PSG population was involved in the expression of fertilin .
Immunocytochemical Localization of Fertilin  Antigenic Sites in PSG and Spermatogonia An immunocytochemical approach was used to determine whether PSG present in the testicular cords of 4-day-old rats (Fig. 5A,B) and in cocultures prepared from testes of 4-day-old rats (Fig. 5D–H) expressed fertilin  protein. Figure 5A shows that
Fig. 2. Developmental expression of selected ADAMs in spermatogonia–Sertoli cell cocultures prepared from 9-day-old rats and cocultured for 24, 48, and 72 hr. Note that cyritestin/ADAM3, ADAM5, fertilin ␣ and , regarded as spermatogenic cell-specific disintegrins, and ADAM6 and ADAM15, are expressed during coculture. Total RNA from testis donor and cocultured cells were used for reverse transcriptase-polymerase chain reaction. Actin mRNA was used as positive control. Marker and amplicon values are indicated in kilobases (kb) and base pairs (bp), respectively. ADAM, A Disintegrin And Metalloprotease.
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Fig. 3. Expression of disintegrins ADAM5, ADAM6, and fertilin ␣ and  in cocultures established from 18-day-old rat testes (containing spermatogonia and primary spermatocytes). The expression of these ADAMs persisted for 72 hr as observed in cocultures prepared from testes of 9-day-old rats shown in Figure 2. Actin mRNA was used as positive control. Marker and amplicon values are indicated in kilobases (kb) and base pairs (bp), respectively. ADAM, a disintegrin and metalloprotease.
the cytoplasm of three large PSG present in the core of the testicular cords displays fertilin  immunoreactivity. Adjacent Sertoli cells do not display immunoreactivity. Omission of anti–fertilin  immunoglobulin (Ig) G resulted in a lack of specific immunoreactivity (Fig. 5B). PSG and Sertoli cell identification was facilitated by the propidium iodide nucleic acid staining patterns in the relatively thin plastic-embedded specimen (1 m thick). PSG display two distinguishing features: a large propidium iodide– stained nucleolus and dispersed
chromatin. Figure 5C shows that large PSG are associated with spherical cells of smaller diameter. Figure 5D illustrates the presence of MitoTracker-stained mitochondria in the cytoplasm of PSG. Abundant mitochondria are another distinctive feature of PSG (McGuinness and Orth, 1992). Figure 5E demonstrates that fertilin  immunoreactivity can be detected in both large PSG (average diameter: 22 m ⫾ 0.3) and smaller spermatogonia (average diameter: 10 m ⫾ 0.4). Figure 5F illustrates merged images of the Mito-
Tracker and anti–fertilin  staining. PSG cocultured with Sertoli cells (Fig. 5G) and immunoreacted with affinity purified anti–fertilin  IgG previously absorbed with recombinant fusion protein (control; Fig. 5H) did not yield the typical staining pattern observed in Figure 5A,E. The characteristic large nucleoli of the cocultured PSG can be clearly seen with propidium iodide staining (Fig. 5I). When PSG–Sertoli cell cocultures were maintained for up to 72 hr, members of the spermatogonial cell cohort derived from PSG precursors, in-
Fig. 4. In situ hybridization of fertilin  in whole testes (days 1 to 7 postnatal) and in prespermatogonia–Sertoli cell cocultures prepared from testes of 4-day-old rats. A: An intense blue color, reflecting fertilin  hybridization of an antisense probe (see Experimental Procedures section), was observed in postnatal testis days 1 to 7. A light blue reaction was observed when a sense oligonucleotide probe was used in a 2-day-old testis, and no color reaction was detected when no oligonucleotide probe was applied (4-day-old testis). B: Prespermatogonia cocultured with Sertoli cells for 24 hr (prepared from 4-day-old testes) do not yield a color signal when no oligonucleotide probe was used (in correlation with the 4-day testis control shown in A). C: An antisense oligonucleotide probe to fertilin  generates a color reaction in selected prespermatogonia (arrow) in contrast with an absent color reaction in adjacent spermatogenic cells (arrowhead). Sertoli cells (SC) in the background are unstained (internal negative control). Fig. 5. Immunocytochemical localization of fertilin  antigenic sites in testicular cords (4-day-old testis) and in prespermatogonia (PSG) cocultured with Sertoli cells (prepared from 4-day-old testis). A: Fertilin  antigenic sites are detected in the cytoplasm of PSG (green staining) still present in the center of the testicular cord. B: A control preparation (absence of primary antibody) does not show specific immunoreactivity. The red propidium iodide staining indicates nucleic acid sites (RNA and DNA). C–F: In this sequence, C shows a phase-contrast microscopic view of two large PSG associated with smaller cells (spermatogonia, arrowheads). D displays the same cell aggregate stained with MitoTracker to detect mitochondria (green staining; arrowheads), a characteristic organelle of PSG and derived cells. E shows the same cluster immunoreacted with anti–fertilin  (red staining). Note that the cells identified as PSG are strongly immunoreactive. Specific immunoreactivity is also detected in some of the spermatogonial cell (arrowheads). Panel F is the merge image of panels D and E. The yellow color indicates colocalization of mitochondria and fertilin  immunoreactive sites, predominantly in PSG. G: A phase contrast microscopy view of two PSG attached to Sertoli cell (SC) surfaces. Each PSG displays a characteristic nucleolus (nu). H: The corresponding view of the specimen immunoreacted with affinity purified anti–fertilin  preabsorbed with recombinant fusion protein. A slight green background staining indicates that the staining with the nonabsorbed affinity purified antibody shown in A and E is specific for fertilin . I: The same specimen stained with propidium iodide (red staining; nu, nucleolus). J: Coculture of spermatogonia and Sertoli cells prepared from 9-day-old rat testes and cocultured for 6 days before staining with anti–fertilin . The arrows indicate remnant PSG surrounded by spermatogonia cells presumably derived from PSG ancestors present at the time of plating. Specific fertilin  immunoreactivity of PSG (green staining, arrows) and derived spermatogonial cells (Spg, identified by arrowheads) is detected. Other spermatogonial cells present in the field are moderately immunoreactive. Sertoli cells in the background (out of focus) are not immunoreactive. Scale bars ⫽ 20 m in A,B,C (applies to C–F),G (applies to G–I), J.
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Fig. 4.
Fig. 5.
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creased in number, formed aggregates of interconnected cells, and displayed fertilin  immunoreactivity (green fluorescence, Fig. 5J). We concluded that PSG express fertilin  protein in the testicular cords and upon PSG culturing and that derived spermatogonial cells continued to express fertilin .
DISCUSSION Data presented in this study demonstrate that several ADAMs regarded to be primarily testis-specific are expressed in fetal rat testis (day 17) and continue to be expressed in the postnatal and adult testis. An unexpected finding was the expression of fertilin  mRNA 2 days after birth, 1 day after the expression of fertilin ␣. As a result, spermatogenic cell-specific fertilin ␣ and  transcripts coexist 48 hr after birth. The early developmental expression of fertilin ␣ (postnatal day 1 rat testis) was previously reported (Frayne et al., 1997). In contrast, the expression of fertilin  mRNA was shown to occur by postnatal day 19 (Frayne et al., 1997), in correlation with the establishment of spermiogenesis (Wolfsberg et al., 1995). We have confirmed the identity of the cloned ADAM species by sequencing the RT-PCR products. Our in situ hybridization and immunocytochemical results indicate that fertilin  transcript and protein are detected in PSG. Further support for the early postnatal expression of fertilin  gene is provided by (1) the demonstration of fertilin  immunoreactivity in PSG cocultured with Sertoli cells (established from 4-day-old testes), (2) the expression of fertilin  mRNA in Sertoli–PSG cocultures prepared from 9-day-old rats (determined by in situ hybridization), and (3) the presence of fertilin  immunoreactivity in cultured spermatogonial cells derived from PSG precursors (prepared from 9-day-old rat testes). Furthermore, our RT-PCR data indicate that fertilin , cyritestin/ADAM3, ADAM5, and ADAM15 mRNAs are expressed before complete spermatogenesis is established. Although we have shown by immunocytochemistry that fertilin  protein is detected in PGC in testis cords and in cocultures, we have
not determined whether fertilin ␣ protein is also expressed and whether a fertilin ␣/ protein heterodimer is present. Our data support the view that gene expression of several ADAMs may be developmentally regulated and that the expression of fertilin  transcripts is restricted to the PSG lineage and derived spermatogonial cell cohorts. The expression and distribution site of fertilin  transcripts in spermatocytes and spermatids have been documented previously (Blobel et al., 1990; Phelps et al., 1990; Wolfsberg et al., 1995). An important aspect that merits further discussion is the functional significance of the early postnatal expression of both fertilin ␣ and  transcripts. Both are synthesized as precursors by spermatogenic cells and processed before sperm maturation (Blobel et al., 1990; Phelps et al., 1990). Both precursors display a multidomain structure characteristic of all members of the disintegrin family: pro-, metalloprotease, disintegrin, cysteine-rich, EGF-like, transmembrane, and cytoplasmic domains (Wolfsberg et al., 1995). A proteolytic processing mechanism removes the pro- and metalloprotease domains, displaying the disintegrin domain at the N-terminus of the fertilin ␣ and  (Blobel et al., 1992; Lum and Blobel, 1997). Peptides containing the disintegrin domain are known to bind to ␣II3 integrin present in platelets (Adler et al., 1991) and fertilin  has been shown to bind to ␣61 integrin present on the egg plasma membrane (Almeida et al., 1995). ␣31 and ␣61 integrins are expressed by both testicular somatic and spermatogenic cells (Schaller et al., 1993; Salanova et al., 1995) during fetal and postnatal testicular development (Frojdman and Pelliniemi, 1994) and in Sertoli–PSG cocultures (Rosselot et al., 2001). Therefore, it is possible that the disintegrin domain of fertilin  may bind to integrins on Sertoli and PSG cell surfaces to modulate PSG–Sertoli cell adhesion during PSG migration to the basal region of the testicular cords. It is noteworthy that, in addition to fertilin  transcripts, cyritestin/ ADAM3, ADAM6, and ADAM15 are
also expressed in fetal and postnatal testis. In contrast to ADAM6 and ADAM15, which are widely expressed (Frayne et al., 1997), both fertilin  and cyritestin/ADAM3 are spermatogenic cell-specific (Heinlein et al., 1994), are expressed as a large precursor (Linder et al., 1995; Yuan et al., 1997), and each has a potential integrin ligand binding site in the disintegrin domain. Peptide mimetics as inhibitors and antibodies directed to active binding sites of fertilin  and cyritestin/ADAM3 were shown to inhibit sperm– egg binding (Yuan et al., 1997; Linder and Heinlein, 1997). Experimental evaluation of the ADAM–integrin role in cell– cell adhesion can also be explored by using the PSG–Sertoli cell coculture system. Cocultures prepared from wild-type and null mutant mice (fertilin -/- [Cho et al., 1998] and cyritestin-/- [Nishimura et al., 2001]) can offer attractive experimental conditions for testing the functional role of fertilin  and cyritestin. Reported data from null mutants indicate that both fertilin  (Cho et al., 1998) and cyritestin (Nishimura et al., 2001) have key roles in sperm plasma membrane adhesion. Mice lacking fertilin  also display a reduction of both fertilin ␣ and cyritestin precursors (Cho et al., 1998). Fertilin ␣ is presumably degraded if it is unable to bind to fertilin  to form a heterodimer (Cho et al., 1998). In the cyritestin null mice, the expression of both cyritestin and fertilin ␣ is absent and fertilin  gene expression is reduced when compared with the wild-type mouse (Cho et al., 1998). Immunopurification data show that the fertilin ␣/ heterodimer and cyritestin are not associated with each other (Cho et al., 1998). Therefore, the independent decrease of cyritestin and fertilin ␣ gene products in the null mutant suggests the existence of a developmental cascade, which may coordinate the temporal synthesis of specific ADAMs required for cell– cell adhesion. In this regard, the coincident gene expression spermatogenic cell-specific fertilin  and cyritestin suggests that more than one ADAM may be required for the modulation of cell– cell adhesion during the migration of PSG from the center to the periphery
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of the testicular cord. Sertoli cell– PGC cocultures could provide an adequate system for approaching experimentally an analysis of cell– cell adhesiveness involving ADAMs and integrins.
EXPERIMENTAL PROCEDURES RT-PCR Total RNA was isolated from rat testis (Sprague-Dawley, Charles River Laboratories) and spermatogonia–Sertoli cocultures using TRI reagent (Molecular Research Center, Cincinnati, OH). Total RNA (1–5 g) was used as a template for first-strand cDNA synthesis using oligo (dT) as a primer, in the presence of Superscript II RNase H- reverse transcriptase (Life Technologies, GIBCO-BRL, Gaithersburg, MD). One l of cDNA was used as a template for the PCR reaction with the following primers within the disintegrin-like domain: rat fertilin , 5⬘ATC AGG GAA AAT GGG CAC CT-3⬘ and 5⬘-AGG TTT GCT TGA ATC AAG AG-3⬘; cyritestin/ADAM3, 5⬘-GGC CCA GCA GAG AGA TGT TC-3⬘ and 5⬘-CAC TGG TCC GTA CTT TTC CTA3⬘; ADAM 5, 5⬘-GAC CGC AGC ACA TTT CAT ACT TAC-3⬘ and 5⬘-CAT GGG GTC ACA GCA TTT TT-3⬘; ADAM15, 5⬘-GGC TCA CAT AAG GCA TGT TAC-3⬘ and 5⬘-ATA TGT TCT GGA TCG GTC TGC-3⬘; and ADAM6, 5⬘GGC TTC CCA GAT GAG TGC AC-3⬘ and 5⬘-CAA TCG TCT GTG GGA AGG CG-3⬘. For rat fertilin ␣, the forward primer was within the metalloproteinase-like domain 5⬘-TCT CGA TGG TGC CTG TTC AG-3⬘ and the reverse primer was from the disintegrin-like domain 5⬘-TGC TTA TCA GGG TTC TTA CA-3⬘ (Frayne et al., 1997). As an internal control, -actin–specific primers were coamplified with ADAM primers to ensure equal cDNA concentrations in comparative studies. PCR parameters used were denaturation at 94°C for 2 min, annealing at 58°C for 10 sec, and extension at 72°C for 30 sec, for 40 cycles. Amplified products were separated on a 1.6% agarose gel using a 1-kb DNA ladder (GIBCO-BRL, Gaithersburg, MD) for size comparison.
Detection of Fertilin  in Rat Testis and Sertoli–Spermatogonia Cell Cocultures by In Situ Hybridization Testis from 1- to 7-day-old rats were fixed for 4 hr in 4% (w/v) buffered formaldehyde and processed for in situ hybridization by using a nonradioactive detection methods as previously described (Hogan et al., 1994). Digoxigenin (DIG) -labeled riboprobes were generated with the DIG RNA labeling kit (Roche Diagnostic Corporation, Indianapolis, IN) from linearized pGEM-3Z-fertilin  plasmid (Promega, Madison, WI). Sense and antisense cRNA probes were synthesized from residues 11871721 of fertilin  cDNA (including the disintegrin domain and part of cysteine-rich domain; GenBank accession no. NM020077). The sense probe was transcribed with T7 RNA polymerase and the antisense probe with SP6 RNA polymerase. Testes were treated with 20 g/ml of proteinase K in PBS for 45 min at 37°C, hybridized with the riboprobes at 60°C for 16 hr, and washed with PIPES buffer (10 mM PIPES, pH 6.8, 1 mM ethylenediaminetetraacetic acid [EDTA], 300 mM NaCl, 1% sodium dodecyl sulfate [SDS]) at 63°C. The samples were incubated with a blocking buffer (100 mM Tris, 150 mM NaCl, pH 7.5, and 2% blocking reagent; Roche) for 2 hr at room temperature before the addition of an alkaline phosphatase-conjugated anti-DIG antibody. After incubation with the antibody (1:2,000 in 2% blocking reagent, 100 mM Tris, 150 mM NaCl, pH 7.5) at 4°C overnight, testes were washed six times with Tris buffer for 1 hr each at room temperature. The samples were equilibrated in alkaline phosphatase buffer (100 mM NaCl, 100 mM Tris, pH 9.5, 50 mM MgCl2, 0.1% Tween 20, and 2 mM levamisole) before the addition of BM Purple AP enzyme substrate (Roche). After 70 –90 min, the color reaction was terminated with 10 mM Tris, 1 mM EDTA, pH 8.0, washed, and photographed using a digital camera. No color reaction
was detected when the probes or antibody conjugate were omitted from the procedure, or when BM Purple AP alone was used as substrate for alkaline phosphatase. Sertoli–spermatogenic cell cocultures were prepared from 4- to 9-day-old rats as previously described (Tres and Kierszenbaum, 1999). For in situ hybridization, cocultures prepared on glass coverslips were fixed with 3.7% (w/v) formaldehyde in 0.1 M sucrose in phosphate buffer (pH 7.0) for 15 min at room temperature, dehydrated through ethanol gradient, and stored until used. Sense (negative control) and antisense oligonucleotides (similar to those used for in situ hybridization of whole testes) were labeled with DIG. Coculture samples were treated with 0.2 N HCl for 20 min at room temperature, heat denatured with 2⫻ SSC (0.015 M sodium citrate, 0.15 M NaCl, pH 7.0) at 70°C for 15 min, washed with phosphate buffered saline, and post-fixed in 4% formaldehyde for 15 min according to a reported procedure for cultured cells (Wilkinson, 1993; Hogan et al., 1994). Hybridization was performed overnight at 37°C, washed successively with PIPES buffer (10 mM PIPES, pH: 6.8, 1 mM EDTA, 300 mM NaCl, 1% SDS) at 63°C. Coverslips were incubated with blocking buffer (100 mM Tris, 150 mM NaCl, pH, 7.5, and 2% blocking reagent; Roche) before the addition of an alkaline phosphatase-conjugated anti-DIG antibody. After incubation with secondary antibody (1:4,000 in 2% blocking reagent, 100 mM Tris, 150 mM NaCl, pH: 7.5) at 4°C overnight, the samples were washed six times with Tris buffer for 20 min each at room temperature. The cells were equilibrated in alkaline phosphatase NTMT buffer (100 mM NaCl, 100 mM Tris, pH 9.5, 50 mM MgCl2, 0.1% Tween 20 and 2 mM levamisole) before the addition of BM Purple AP enzyme substrate (Roche). After 90 min, the color reaction was terminated with 10 mM Tris, 1 mM EDTA, pH 8.0, washed, and photographed with a Magnafire digital camera (Optronics, Bolton, MA).
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Detection of Fertilin  in Rat Testis and Sertoli–Spermatogonia Cell Cocultures by Indirect Immunofluorescence For immunodetection of fertilin  in rat testis, small minces (approximately 1–2 mm3) of testis (collected from 4-day-old rats) were fixed in a mixture of 1.5% glutaraldehyde and 3.4% paraformaldehyde (electron microscope grade) in 0.1 M phosphate buffer, pH 7.2, and embedded in Lowicryl K4M (Polysciences, Warrington, PA) as described (Rivkin et al., 1997). Samples were sectioned by using glass knives, and 1-m-thick sections of Lowicryl-embedded testis were placed on microscope slides and processed for indirect immunofluorescence. Samples were blocked with goat serum and incubated overnight at 4°C with affinity purified rabbit polyclonal anti–fertilin  IgG against rabbit ADAM-2 (a generous gift from Dr. Chris M. Hardy, CSIRO, Lyneham, Australia [Hardy and Holland, 1996]; working dilution:1:50 in 0.1 M sucrose containing 0.1% Tween-20, 0.1% gelatin, and 0.2% bovine serum albumin). Samples were rinsed with deionized water and reacted with secondary antibody (goat anti-rabbit IgG conjugated with fluorescein isothiocyanate or tetramethylrhodamine isothiocyanate [TRICT] from Jackson Immunochemicals, West Grove, PA) using a working dilution of 1:200 for 1 hr at room temperature. Controls included absorption of anti–fertilin  using recombinant fusion protein (a gift from Dr. Hardy) and omission of primary IgG. Samples were rinsed and mounted with Vectashield containing propidium iodide to stain nucleic acids (Vector Laboratories). Air-dried preparations of Sertoli–spermatogenic cell cocultures prepared on glass coverslips were rehydrated in PBS for 15 min and processed for indirect immunofluorescence as described above for Lowicryl-embedded sections. MitoTracker (Molecular Probes, Eugene, OR) was used to detect mito-
chondria in PSG as recommended by the manufacturer.
ACKNOWLEDGMENT L.L.T. received funding from the NIH.
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