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Testis |
Department of Anatomy and Cell Biology,3 The University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3
Department of Biology,4 Chiba University, Chiba 263-8522, Japan
Department of Molecular Biosciences,5 Washington State University, Pullman, Washington 99164-4660
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sertoli cells, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Three possible functions have been proposed for tubulobulbar complexes. The first is that tubulobulbar complexes function to attach the mature spermatids to Sertoli cells [1, 3, 4]. The second is that these structures remove excess spermatid cytoplasm and acrosomal contents prior to spermatid release [4, 5]; the final possible function is that the structures internalize junction domains in the plasma membrane [3, 6].
Because actin is a major component of ectoplasmic specializations, numerous studies have focused on identifying actin-associated components at ectoplasmic specializations [7–13]. Recently, the calcium-dependent actin-severing and -capping protein gelsolin also has been identified at ectoplasmic specializations [12]. This protein is thought to function in the disassembly of the actin layer of the junction plaques. Based on this finding, we were interested to see if a calcium-independent actin-severing protein also was found at these sites.
Cofilin is a calcium-independent actin-depolymerizing protein, which disassembles actin filaments when unphosphorylated. Two isoforms of cofilin (muscle type and non-muscle type) exist, and mRNA of both types have been found in the testis [14]. There are two groups of proteins that have been identified thus far that have the capability of phosphorylating cofilin and rendering it inactive. These proteins are the LIM kinases (LIMKs) and the testicular protein kinases (TESKs), respectively. LIMK2 null male mice exhibit impaired spermatogenesis [15]. Interestingly, with all of this data implicating cofilin as an important factor in spermatogenesis, the protein has still not been localized to any specific structures in the testis.
In this study we used a pan-cofilin antibody to immunolocalize cofilin in the seminiferous epithelium. We found that this probe specifically labeled tubulobulbar complexes in the region occupied by filamentous actin. It did not label ectoplasmic specializations, the only other major site of actin filament concentration in Sertoli cells. On two-dimensional (2D) Western blots of rat seminiferous epithelium, only two, rather than four, protein dots appeared, which would be predicted due to the two cofilin isoforms (muscle type and non-muscle type) and their ability to be phosphorylated and dephosphorylated. Microarray analysis indicated that the message for non-muscle type cofilin is 8.5-fold greater than for the muscle type isoform in Sertoli cells. Antibodies raised specifically against non-muscle type cofilin labeled tubulobulbar complexes and reacted with the same two dots that reacted with the pan-cofilin antibody on 2D rat seminiferous epithelium Western blots. The data presented here support the finding that the non-muscle cofilin isoform is present at tubulobulbar complexes and that neither the muscle or non-muscle cofilin isoforms are concentrated at ectoplasmic specializations.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All animals used in these studies were reproductively active male Sprague Dawley rats. They were obtained from the University of British Columbia animal care colony and were maintained according to the guidelines established by the Canadian Council on Animal Care. Microarray studies were performed using mice from Washington State University. Their use conformed to the protocols approved by the Washington State University Animal Care and Use Committee and the National Institutes of Health standards detailed in the Guidelines for the Care and Use of Experimental Animals.
Chemicals and Reagents
Unless otherwise indicated, all chemicals and reagents were obtained from Sigma Aldrich Canada (Mississauga, ON, Canada). The paraformaldehyde and NaCl were obtained from Fisher Scientific (Vancouver, BC, Canada). All control immunoglobulins (IgG) as well as all secondary antibodies conjugated to horseradish peroxidase were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Secondary antibodies conjugated to ALEXA fluorochromes were all purchased from Molecular Probes (Eugene, OR).
Immunofluorescence
Tissue preparation Testes were removed from male rats under deep anesthesia. Warm (33°C) PBS (150 mM NaCl, 5 mM KCl, 0.8 mM KH2PO4, 3.2 mM Na2HPO4, pH 7.3) was perfused through the spermatic artery using a 26-gauge needle attached to a gravity-fed perfusion apparatus for 2 min to clear the organ of blood. Following this, warm 3% paraformaldehyde in PBS was perfused through the testis for 30 min. PBS was then reperfused through the organ to wash out any remaining fixative.
Frozen sections For frozen sections, the fixed testes were frozen (using liquid nitrogen) while at the same time being attached to an aluminum stub by OCT compound (Sakura Finetek USA, Inc., Torrance, CA). Frozen testis sections were cut, attached to poly-l-lysine-coated glass slides, immediately plunged into -20°C acetone for 5 min, allowed to air dry, and then processed for immunofluorescence.
Fragmented material For fragmented material, perfusion-fixed testes were decapsulated, cut into 1-mm sized cubes, and transferred into a 15-ml plastic Falcon tube along with about 5 ml of PBS. The material was gently passed through an 18-gauge needle followed by a 21-gauge needle for two to five gentle passes. This fragmented material was left to sediment by gravity at room temperature for 10–15 min, at which point the uppermost layer was added to poly-l-lysine-coated slides and allowed to incubate in a humidity chamber for 10 min. All excess PBS was then removed and slides were immediately treated with -20°C acetone for 5 min and allowed to air dry, at which point 5% blocking serum was added.
Antibody labeling Once the tissue was ready for serum blocking, 5% normal goat serum (NGS) in TPBS-BSA (PBS containing 0.05% Tween-20 and 0.1% bovine serum albumin) from the secondary antibody host was incubated on the testis material for 20 min at room temperature. This was used to block the tissue from secondary antibody nonspecific binding. The primary antibodies (consisting of a mouse monoclonal anticofilin Mab-22 [16] and a rabbit serum anti-non-muscle cofilin [17]) were added to the experimental slides made up in TPBS-BSA with 1% NGS and incubated overnight at 4°C in a humidity chamber. The material was washed extensively with the TPBS-BSA (wash buffer) and then incubated for 60 min at 37°C with secondary antibody conjugated to a fluorochrome (goat anti-mouse ALEXA 488 or goat anti-rabbit ALEXA 568). After further washing, coverslips were mounted using Vectashield (Vector Labs, Burlington, ON, Canada) and visualized by standard fluorescence microscopy using a Zeiss Axiophot microscope (Carl Zeiss, Oberkochen, Germany).
Filamentous actin labeling was performed using ALEXA 488 or ALEXA 568 phalloidin (Molecular Probes).
Controls consisted of replacing the primary antibody with its respective IgG or serum at identical concentrations to the primary antibody, replacing the primary antibody with buffer alone, or replacing both the primary and secondary antibodies with buffer alone.
1D Western Blotting
Testis material was loaded into wells of 1-mm-thick 10% SDS-PAGE gels and run according to standard protocols [18]. Proteins were transferred onto Immobilon-P transfer membrane (Millipore, Watertown, MA), then washed for 5 min at room temperature with TBST (500 mM Tris pH 7.5, 150 mM NaCl, 0.1% Tween-20). The blots were then blocked for 8 h at 4°C using 4% nonfat milk (Blotto, Santa Cruz Biotechnology, Santa Cruz, CA) in order to decrease nonspecific antibody binding. Following blocking, membranes were washed three times for 10 min each, then incubated with the mouse anti-cofilin Mab-22 or rabbit anti-non-muscle cofilin antibody overnight at 4°C. The following day, blots were washed extensively with TBST followed by a 1-h incubation with the secondary antibody (goat anti-mouse or goat anti-rabbit conjugated to horseradish peroxidase; Jackson ImmunoResearch) at room temperature. Upon further washing with TBST followed by TBS, blots were reacted with ECL (Pharmacia-Pfizer, New York, NY) to visualize the reactive bands on X-OMAT film (Kodak, Rochester, NY).
Controls consisted of replacing the primary antibodies with normal mouse IgG or normal rabbit serum at identical primary antibody concentrations or identical serum dilutions.
2D Electrophoresis
Isolated rat seminiferous epithelium, in solution, was first centrifuged at 10 000 rpm using an Eppendorf (Hamburg, Germany) desktop centrifuge for 15 min at room temperature. Once completed, the supernatant was collected and treated to acetone precipitation. To do this, three volumes of -20°C acetone was added to the sample and incubated at -20°C for 30 min with occasional agitation. The sample proteins were then pelleted, and the supernatant was discarded. The resulting protein pellet was allowed to air dry for 10–15 min and was resuspended in 2D-gel sample buffer (8 M urea, 2 M thiourea, 4% w/v CHAPS, 20 mM Tris, 0.0025% bromophenol blue) at room temperature on a Labquake rotator (Barnstead International, Dubuque, IA) for 6–8 h. Once resuspended, 0.5% of immobilized pH gradient (IPG) buffer (Amersham, Quebec, QC, Canada) at the same pH as the IPG focusing strip was added to the sample. After a 5-min centrifugation at 10 000 x g in an Eppendorf desktop centrifuge, the remaining supernatant was added to the IPG strip for a 24-h strip rehydration at 15°C. Focusing was then performed using the IPGphor isoelectric focusing machine (Amersham). Focused IPG strips were loaded onto large-format slab gels (Bio-Rad) and run at 50–85 V.
Microarray Analysis
Mouse Sertoli cells were isolated from whole testes from 14- to 16-day-old Rosa and B6/129 mice and cultured using standard culture procedures in DMEM/F12 medium following the procedure of Karl and Griswold [19]. Cells were incubated at 32°C in 5% CO2 for 2 days following isolation. The isolation of Sertoli cells was performed on two independent occasions to provide duplicate samples for microarray analysis.
Total RNA from the enriched Sertoli cell culture was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA) following the manufacturer's instructions. The quality and concentration of the purified RNA was determined using absorbencies at 260 and 280 nm. The standard Affymetrix (Santa Clara, CA) protocol required 10 µg of total RNA with a minimum 260:280 ratio of 1.80.
Ten micrograms of total RNA was used to synthesize the Affymetrix GeneChip microarray target. Briefly, double-stranded cDNA was synthesized from the original RNA using reverse transcription with an oligo-(dT17) primer followed by RNase A treatment and DNA polymerase synthesis of the second strand. The double-strand cDNA was used as a template for an in vitro transcription reaction using biotinylated cytosine 5'-triphosphate and uridine 5'-triphosphate to produce labeled cRNA (MegaScript, Ambion, Austin, TX). The biotinylated cRNA was then fragmented and hybridized to the MGU74Av2 GeneChip arrays (Affymetrix). The microarrays were processed with Affymetrix GeneChip Fluidics Workstation 400 using the Mini-Euk 2v3 protocol, stained with phycoerythrin-coupled streptavidin, and scanned on a Hewlett-Packard gene array scanner (Hewlett-Packard Co., Palo Alto, CA). Microarray data were scaled to a target signal of 125 and analyzed with Microarray Suite 5.0 software (Affymetrix).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To identify which isoform of cofilin may be present specifically in Sertoli cells, the region where the filamentous actin surrounds the tubulobulbar complex, we utilized GeneChip microarrays (Affymetrix) to quantify the amount of muscle and non-muscle type cofilin transcript found in an enriched murine Sertoli cell population. Transcript abundances of 1905 and 219 were detected for non-muscle type cofilin and muscle type cofilin, respectively, representing an over 8.5-fold increase of non-muscle type cofilin mRNA compared with muscle type.
Although not always reflective of protein abundance, the mRNA data led us to use a previously characterized rabbit serum non-muscle type cofilin antibody [14] for immunolocalization on fixed, fragmented testis material. Using this antibody, we found specific labeling at tubulobulbar complexes (Fig. 5a) as well as nonspecific staining along the dorsal curve of the spermatid head (Fig. 5b). This well-known, nonspecific staining pattern often is seen when using rabbit antibodies on testis material. A protein at 20 kDa was clearly labeled by 1D Western blots (Fig. 6a), as well as other higher molecular weight non-specific bands that were accounted for by the normal rabbit serum primary antibody control blot (Fig. 6b). To see if the two reactive protein dots on the pan-cofilin (Mab-22) 2D Western blot were in fact the non-muscle isoform of cofilin, we again performed 2D Western blots of isolated rat seminiferous epithelium and probed them with the non-muscle type cofilin antibody. As predicted, the same two reactive protein dots appeared in the appropriate location for cofilin (Fig. 6c).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cofilin is a calcium-independent actin filament regulatory protein. To date, two isoforms (muscle type and non-muscle type) have been discovered in mammals, and the isoforms are encoded by different genes. Messenger RNA transcripts for muscle type cofilin have been found in skeletal muscle, heart, and testis, whereas mRNA transcripts for non-muscle cofilin have been found in brain, lung, spleen, and testis [14].
Actin filaments in Sertoli cells are concentrated at tubulobulbar complexes and at ectoplasmic specializations, both of which develop at sites of intercellular attachment. Actin filaments in tubulobulbar complexes occur as a network, whereas those in ectoplasmic specializations are unipolar and hexagonally packed into bundles. A cistern of endoplasmic reticulum, which is a potential calcium store, is directly associated with filament bundles in ectoplasmic specializations. A similar cistern of endoplasmic reticulum is not found in association with the filament networks in tubulobulbar complexes. The presence of cofilin, a calcium-dependent actin filament-severing protein, in tubulobulbar complexes and not at ectoplasmic specializations may be related to differences in local levels of calcium and the way it is regulated at the two sites.
The conclusion that non-muscle cofilin is the isoform of the protein predominantly expressed in the seminiferous epithelium and at tubulobulbar complexes is supported by three observations. First, there is an 8.5-fold higher abundance of non-muscle type cofilin mRNA over muscle type cofilin mRNA in Sertoli cells. Second, immunoblots probed with the pan-cofilin Mab-22 antibody indicate that mainly one isoform is expressed in the seminiferous epithelium. Third, staining with an antibody specific for non-muscle cofilin labeled tubulobulbar complexes in sections and the same protein as the Mab-22 antibody on immunoblots of seminiferous epithelium. Based on these findings we conclude that the isoform of cofilin at tubulobulbar complexes is likely the non-muscle type. Although we focused on apical tubulobulbar complexes in this study, we anticipate that cofilin also will be found to be a component of basal complexes once these structures are fully investigated.
Because both non-muscle type cofilin and gelsolin (personal observation) are present at tubulobulbar complexes, the two proteins probably act together and in concert with other actin-binding proteins to control actin filament dynamics at tubulobulbar complexes. Although non-muscle cofilin is specifically concentrated at tubulobulbar complexes, its precise function at these sites remains to be experimentally verified, although a role in disassembly of actin filaments during cell detachment is likely.
Cofilin previously has been reported in mouse spermatogenic cells [15], where it is diffusely localized in the cytoplasm. However, we were unable to convincingly demonstrate differences in staining between spermatogenic cells in antibody and control treated normal rat tissue sections in our study. Differences in results between our study and the previous study may be due to differences in tissue handling or to species differences in reactivity. Also, the diffuse nature of cofilin staining in spermatogenic cells may have been below what we could convincingly detect using our techniques. Perhaps the strongest evidence for the presence of the protein in spermatogenic cells is that testicular fractions (mouse) enriched for spermatocytes react positively on immunoblots treated with cofilin antibodies [15]. In addition, nuclear inclusions of cofilin occur in spermatocytes of LIMK null mice after the testes have been exposed to experimental hyperthermia [15].
LIMK specifically catalyzes the phosphorylation of cofilin [20–22]. There are two LIMK genes that have been described to date in mouse, rat, and human—LIMK1 and LIMK2 [23–25]. LIMK1 is thought to be specifically activated by the small GTPase rac, whereas LIMK2 is thought to be activated by the rho and cdc42 G-proteins [22, 26]. The finding of impaired spermatogenesis and increased apoptosis of spermatogenic cells in LIMK2 null mice is consistent with the conclusion that LIMK2 is important in male reproduction presumably by regulating either the activity of cofilin or its distribution in spermatogenic cells [15].
Although LIMK2 has been demonstrated in spermatogenic cells [15], neither isoform of LIMK has been successfully localized in Sertoli cells. It is possible that phosphorylation of cofilin in Sertoli cells is regulated by proteins other than LIMK. Among possible candidates are the TESK proteins.
In contrast to the LIMKs, the TESK proteins (LIMK-like proteins) do not appear to be activated by small GTPases; rather, they appear to be activated by integrins [27]. Interestingly, the ß1 integrin is a component both of ectoplasmic specializations and of tubulobulbar complexes [28, 29]. Thus far two TESK proteins have been described, TESK1 and TESK2. TESK1 is found primarily in testicular germ cells, whereas TESK2 primarily is found in Sertoli cells. Although the function of TESK2 in the testis has not been determined, it has been shown to phosphorylate cofilin in vitro [27]. This finding together with the observations that TESK2 is primarily expressed in Sertoli cells and is likely activated through an integrin-based mechanism make TESK2 a prime candidate for regulating the phosphorylation, and therefore activity, of cofilin.
Based on our results from immunofluorescence and immunoblots, we conclude that non-muscle cofilin is a component of tubulobulbar complexes and is not a significant component of ectoplasmic specializations in rat Sertoli cells. The presence of this protein at tubulobulbar complexes provides a calcium-independent way of controlling actin filament dynamics at these sites.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: A. Wayne Vogl, 313-2177 Wesbrook Mall, The University of British Columbia, Department of Anatomy and Cell Biology, Vancouver, BC, Canada V6T 1Z3. FAX: 604 822 2316; vogl{at}interchange.ubc.ca
Received: 3 September 2003.
First decision: 21 September 2003.
Accepted: 10 November 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-actinin and vinculin with actin: opposite effects in filament network formation. Proc Natl Acad Sci U S A 1981 78:3005-30096ß1 is localized at specific sites of cell-to-cell contact in rat seminiferous epithelium. Biol Reprod 1995 52:79-87[Abstract]This article has been cited by other articles:
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  J. S. Young, J. A. Guttman, K. S. Vaid, H. Shahinian, and A. W. Vogl Cortactin (CTTN), N-WASP (WASL), and Clathrin (CLTC) Are Present at Podosome-Like Tubulobulbar Complexes in the Rat Testis Biol Reprod, January 1, 2009; 80(1): 153 - 161. [Abstract] [Full Text] [PDF]
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S. E Fiedler, M. Bajpai, and D. W Carr Identification and Characterization of RHOA-Interacting Proteins in Bovine Spermatozoa Biol Reprod, January 1, 2008; 78(1): 184 - 192. [Abstract] [Full Text] [PDF]
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K. S Vaid, J. A Guttman, R. R Singaraja, and A. W. Vogl A Kinesin Is Present at Unique Sertoli/Spermatid Adherens Junctions in Rat and Mouse Testes Biol Reprod, December 1, 2007; 77(6): 1037 - 1048. [Abstract] [Full Text] [PDF]
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 H. Obermann, I. Raabe, M. Balvers, B. Brunswig, W. Schulze, and C. Kirchhoff Novel testis-expressed profilin IV associated with acrosome biogenesis and spermatid elongation Mol. Hum. Reprod., January 1, 2005; 11(1): 53 - 64. [Abstract] [Full Text] [PDF]
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 N. P.Y. Lee and C. Y. Cheng Ectoplasmic specialization, a testis-specific cell-cell actin-based adherens junction type: is this a potential target for male contraceptive development? Hum. Reprod. Update, July 1, 2004; 10(4): 349 - 369. [Abstract] [Full Text] [PDF]
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