Zhibing Zhang, Department of Obstetrics/Gynecology, Virginia Commonwealth University, 1101 E Marshall Street, Richmond, VA 23298, USA. E-mail: email@example.com
Spermiogenesis is the final phase of spermatogenesis. During this process, haploid round spermatids differentiate into spermatozoa, with dramatic morphological changes, including elongation and condensation of the nuclei, and formation of the flagella. Meig1 is one of many genes involved in the regulation of this process. Male mice deficient in MEIG1 are sterile with a severe defect in spermiogenesis, associated with dramatic disruption of the spermatid manchette and failure of flagellogenesis. A yeast two-hybrid screen using full-length MEIG1 as bait identified transcription factor-like 5 protein (TCFL5) as a putative interacting proteins. Interestingly, this protein was also identified as a potential binding partner of SPAG16, another protein essential for spermatogenesis, and also a binding partner of MEIG1. The interaction between TCFL5 and MEIG1 was confirmed in cultured cells over-expressing the two proteins. The mouse Tcfl5 transcript is present only in the testis, and its expression is significantly increased during spermiogenesis. However, little is known about TCFL5 protein and its role in male germ cells. A rabbit polyclonal antibody was generated against the C-terminal region of TCFL5. Mouse TCFL5 protein was expressed in the testis but not in mature spermatozoa. During the first wave of spermatogenesis, TCFL5 expression was dramatically increased at day 30 after birth. In the testis and a mixture of dispersed testicular cells, the protein co-localized with α-tubulin, a manchette marker in early elongating spermatids. The protein also localized in the centrioles of late elongating spermatids. No obvious differences in TCFL5 epitope abundance and localization were observed between wild type and the Meig1-deficient mice. These findings suggest that TCFL5 may play a role upstream of MEIG1 action, and based on putative binding partners and localization is likely to be involved in spermiogenesis and formation of the sperm flagella.
Spermatogenesis can be divided into three phases: proliferation of spermatogonia by mitosis, meiotic division of spermatocytes and differentiation of haploid spermatids by spermiogenesis. The whole process takes place in the testis within the seminiferous tubules (Eddy, 2002; Hess & de Franca, 2008). During spermiogenesis, haploid round spermatids differentiate into species-specific-shaped spermatozoa (Hermo et al., 2010a). These final steps consist of dynamic morphological changes such as condensation of the nucleus (Martins & Krawetz, 2007; Chiva et al., 2011), formation of an acrosome and sperm flagellum (Moreno & Alvarado, 2006; Rajender et al., 2010) and reorganization of organelles including the formation of the mitochondrial sheath along the midpiece of the flagellum (Hermo et al., 2010b). The spermatid loses almost all of its cytoplasm (Zheng et al., 2007), resulting in a slender, condensed, mature spermatozoon that is released into the lumen of the seminiferous tubule during spermiation (O'Donnell et al., 2011).
Spermiogenesis is controlled by many genes that have been identified through gene targeting (Yan, 2009). Our previous studies demonstrated that mouse meiosis-expressed gene 1 (Meig1) is a key regulator (Zhang et al., 2009). Meig1 was originally identified in a search for mammalian genes potentially involved in meiosis (Don & Wolgemuth, 1992; Don et al., 1994; Chen-Moses et al., 1997; Ever et al., 1999; Steiner et al., 1999). The message is also present in foetal Sertoli cells in foetal gonads and a Sertoli cell line TTE3 (Tabuchi et al., 2006; Bouma et al., 2007). To study the function of this gene, we inactivated the Meig1 gene and, unexpectedly, found that Meig1 mutant male mice had no obvious defect in meiosis, but were sterile as a result of impaired spermiogenesis. Transmission electron microscopy revealed that the manchette, a microtubular organelle essential in the formation of the sperm head and flagellum was disrupted in spermatids of Meig1-deficient mice (Zhang et al., 2009). These observations further illuminate another report of this phenotype (Salzberg et al., 2010). To identify potential binding partners of MEIG1, a yeast two-hybrid screen was conducted and several potential binding partners were identified, including PACRG (Zhang et al., 2009) and transcription factor-like 5 protein (TCFL5, GenBank accession number: NM_178254).
In our previous study, MEIG1 was identified as a binding partner of sperm-association antigen 16 (SPAG16) (Zhang et al., 2004). Mouse Spag16 is a bi-functional gene that encodes full-length 71 kDa SPAG16L, which is assembled into sperm flagella and regulates sperm motility (Zhang et al., 2006), and 35 kDa SPAG16S, which is also localized in the nuclei of round spermatids and is believed to participate in the regulation of spermatogenesis (Zhang et al., 2004). Both proteins contain seven copies of the WD motif, a conserved domain that mediates protein–protein interactions (Craig, 2003). Interestingly, when the yeast two-hybrid screen was conducted using the WD repeats as bait, besides MEIG1, in the same screen mouse TCFL5 was also identified as another potential binding partner (Zhang et al., 2008). Because TCFL5 appears to interact with the two key players, MEIG1 and SPAG16, we hypothesised that TCFL5 may also play a role in the regulation of spermatogenesis/spermiogenesis. TCFL5 protein was found to be specifically expressed in the testis during steps of spermiogenesis. Although TCFL5 contains a nuclear localization signal, it is localized to the manchette of early elongating spermatids and centrioles of late elongating spermatids; however, the localization was not dependent on the MEIG1 protein. These findings suggest that TCFL5 may play a role in spermiogenesis, possibly in formation of sperm flagellum.
Materials and methods
All rodent work was approved by Virginia Commonwealth University's Institutional Animal Care & Use Committee (protocol permit #AM10297) in concordance with all federal and local regulations regarding the use of non-primate vertebrates in scientific research.
Identification of the Tcfl-5 cDNA
The mouse Tcfl-5 cDNA (NM_178254) was identified from a yeast two-hybrid screen using full-length MEIG1 and WD repeats of mouse SPAG16 protein as baits (Zhang et al., 2008).
Reverse transcription–polymerase chain reaction
Total mouse RNAs from the indicated tissues and the testes at different spermatogenic stages were reversed transcribed using the Transcriptor first-strand cDNA synthesis kit from Roche (Indianapolis, IN, USA). RT-PCR was conducted using cDNAs transcribed from these RNAs to examine the expression of the Tcfl5 transcript with the following primers: forward: 5′-AAGCGTGGAGCGAAGAGCCC-3′; and reverse : 5′-GCAGGTCACCAGGGATTCGGG-3′. Actin, a housekeeping gene, was used as the control.
Mammalian expression constructs
Complementary DNA containing the full-length mouse Tcfl5 cDNA was amplified by RT-PCR using the following primers: forward: 5′-GAATTCTTCGTCTGCGGAGGCCGCGACCCCG-3′; and reverse: 5′-GGATCCTCACTTGATCTCCATGGCAGGGCT-3′. After sequencing, the PCR products were cloned into EcoRI/BamHI sites of the pEGFP-C1 vector, creating the full-length TCFL5/pEGFP-C1 plasmid.
Co-immunoprecipitation from transfected cells
COS-1 cells were co-transfected with TCFL5/pEGFP-C1 and MEIG1/pTarget plasmids. Forty-eight hours later, the cells were harvested into immunoprecipitation buffer (150 mm NaCl; 50 mm Tris·HCl, pH 8.0; 5 mm EDTA; 1% Triton X-100; 1 mm PMSF and proteinase inhibitor mixture), and the lysates were passed through a 20-gauge needle. After centrifugation at 11 600 × g for 5 min, the supernatants were pre-cleared with protein A beads at 4 °C for 30 min. The supernatants were then incubated with 1 μL (1 μg/μL) of anti-MEIG1 antibody or pre-immune serum at 4 °C for 2 h, and protein A beads were added with a further incubation at 4 °C overnight. The beads were washed with immunoprecipitation buffer three or four times; 1× protein loading buffer was then added to the beads, which were boiled at 100 °C for 10 min; and the samples were then processed for western blotting using monoclonal anti-GFP, or anti-MEIG1 polyclonal antibody.
Generation of the anti-TCFL5-specific antibody
A cDNA encoding a C-terminal portion of mouse TCFL5 was amplified by PCR from a Tcfl5 cDNA clone using a forward primer: 5′-CATATGCAAGCAATCAATAAGAGGAATC-3′ (NdeI), and a reverse primer: 5′-GAATTCCTTGATCTCCATGGCAGGGCTG-3′ (EcoRI). The cDNA was inserted into NdeI/EcoRI sites of the pET28a vector (Novagen, Madison, WI, USA). The resulting fusion protein contained His6 tags at both the N and C termini. The construct was transformed into BL-21(DE3) cells, and the fusion protein was induced and subsequently purified as reported previously (Zhang et al., 2005). The purified recombinant protein was used to generate polyclonal antisera in rabbits by a commercial organization (Antibody Research Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences).
Western blot analysis
Equal amounts of protein (50 μg/lane) were heated to 95 °C for 10 min in sample buffer, loaded onto 10% sodium dodecyl sulphate-polyacrylamide gels, electrophoretically separated and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Membranes were blocked [Tris-buffered saline solution containing 5% non-fat dry milk and 0.05% Tween 20 (TBST)] and then incubated with indicated antibodies at 4 °C overnight. After washing in TBST, the blots were incubated with second antibodies for 1 h at room temperature. After washing, the proteins were detected with Super Signal chemiluminescent substrate (Pierce, Rockford, IL, USA). To examine the specificity of the antibody, before incubating the antibody with the membrane, the antibody was mixed with the purified protein which was used to generate the antibody, and western blot was conducted with the neutrolized antibody.
Immunolocalization of TCFL5 in mouse testis
Testes from wild-type adult males were fixed with 4% paraformaldehyde in 0.1 m PBS (pH 7.4), and 5 μm paraffin sections were made. Immunofluorescence was conducted using the method described by Tsuneoka (Tsuneoka et al., 2006). The sections were double stained with the polyclonal anti-TCFL5 (1 : 200) and monoclonal anti-α-tubulin or anti-γ-tubulin antibodies. After incubating with the primary antibodies, the slides were washed with PBS and incubated 1 h at room temperature with Alex 488-conjugated anti-mouse IgG secondary antibody or Cy3-conjugated anti-rabbit IgG secondary antibody. Following secondary antibody incubation, slides were washed in PBS, mounted with VectaMount-DAPI (Vector Laboratories, Burlingame, CA, USA) and sealed with a cover slip. Images were taken by confocal laser-scanning microscopy.
Enzyme-dissociated testicular cell preparations and immunofluorescence
Enzyme-dissociated testicular cells were prepared using a previously described method (Xu et al., 2008). In brief, testes from an adult wild-type mouse were decapsulated, placed in 5 mL DMEM containing 0.5 mg/mL collagenase IV (Sigma-Aldrich, St. Louis, MO, USA) and 1.0 mg/mL DNase I (Sigma-Aldrich), and incubated 30 min at 32 °C. The dissociated testicular cells were then centrifuged 5 min at 200 g. The dispersed mixture of testicular cells was fixed in 4% paraformaldehyde/PBS (containing 4% sucrose) at room temperature for 15 min, then washed three times with PBS. Prior to plating, cells were re-suspended in 12.5 mL PBS, and 100 μL of cell suspension was spread on SuperFrost/Plus microscope slides (Fisher Scientific, Pittsburgh, PA, USA) and allowed to air dry. The cells were permeabilized with 1% Triton X-100 for 5 min at 37 °C, blocked 1 h at room temperature with 10% goat serum in PBS. Following overnight incubation with primary antibodies (diluted in blocking medium at a 1 : 200 dilution for anti-TCFL-5 antibody; 1 : 100 for anti-α-tubulin) at 4 °C, slides were washed with PBS and incubated 1 h at room temperature with the same secondary antibodies as used for tissue sections. The cells were washed three times in PBS, mounted, sealed and images were captured as described above.
Meig1-deficient mice were generated previously (Zhang et al., 2009).
TCFL5 was associated with MEIG1
From our original yeast two-hybrid screen using MEIG1 as bait, TCFL5 was identified to be a potential binding partner. To further study the interaction between the two proteins, TCFL5/pEGFPC1 mammalian expression plasmid was constructed, and the GFP-tagged TCFL5 protein was expressed in the transfected COS-1 cells (Fig. 1A). To explore the interaction of TCFL5 and MEIG1, TCFL5/pEGFPC1 and MEIG1/pTarget plasmids were co-transfected into COS-1 cells, and Co-IP was performed. The cell lysates were pulled down with an anti-MEIG1 antibody, and western blotting was carried out with an anti-MEIG1 antibody and an anti-GFP antibody. MEIG1 antibody not only immunoprecipitated MEIG1 protein (Fig. 1B, upper part), TCFL5/GFP protein was co-immunoprecipitated from the lysate (Fig. 1B, lower part).
Tissue distribution of mouse and human Tcfl5 mRNAs
To determine the tissue distribution of Tcfl5 mRNA, RT-PCR was conducted using cDNAs derived from mouse and human tissues. Mouse Tcfl5 mRNA was expressed only in the testis (Fig. 2A).
Tcfl5 mRNA expression in the testis during the first wave of spermatogenesis
Expression of the mouse Tcfl5 gene was investigated in the testis during the first wave of spermatogenesis by RT-PCR. The message was undetectable from days 6–16 after birth. The message was expressed starting from day 20, increased significantly on day 30 and remained the same level through day 42 post birth (Fig. 2B).
Tissue distribution of TCFL5 protein
A polyclonal antibody was generated against C-terminus of the full-length TCFL5 protein, and western blot was conducted using mouse tissues. The antibody cross-reacted with the 57 kDa TCFL5 protein only in the testis (Fig. 3A). In addition, the antibody also recognized a 37 kDa protein in smooth muscle (Fig. 3A), but was not detected in other tissues examined. The 37 kDa protein in the smooth muscle appeared to be non-specific, as Tcfl5 message was not present in this tissue (Fig. 2A), and the neutralized antibody still recognized this protein, but not the 57 kDa TCFL5 protein (Fig. 3B).
TCFL5 protein expression during the first wave of spermatogenesis
To further examine dynamic expression of TCFL5 protein in the testis during the first wave of spermatogenesis, western blotting was conducted using testicular extracts from mice at indicated ages and spermatozoa collected from cauda epididymis. The 57 kDa protein was expressed from day 20, significantly increased at day 30 after birth and was maintained at a similar level on day 42 (Fig. 4, left part). Even though TCFL5 was highly expressed in testis, it was not present in spermatozoa (Fig. 4, right part).
TCFL5 was localized in the manchette of early elongating spermatids
Endogenous TCFL5 protein localization was examined with testicular sections from adult wild-type mice. The pre-immune serum failed to detect any specific signal in the mouse testis sections (Supplemental Figure S1). No specific immunostaining was observed in spermatogonia and spermatocytes. In early elongating spermatids, staining was largely restricted to the region surrounding the nuclear membrane, and co-localized with α-tubulin, a manchette marker (Fig. 5, Supplemental Figure S2). The sections were also double stained with an anti-γ-tubulin antibody, which recognizes the centriole. TCFL5 did not co-localize with γ-tubulin at this stage (Fig. 6, Supplemental Figure S3). The localization of TCFL5 was further examined using mixed germ cells isolated from wild-type mice. Similar to what was observed in the testicular section, TCFL5 co-localized with α-tubulin (Supplemental Figure S4).
TCFL5 was localized in the centriole of the late elongating spermatid
TCFL5 localization in late elongating spermatids was examined in testicular sections. Unlike the early elongating spermatids, TCFL5 was concentrated near the caudal region of the nucleus, and co-localized with γ-tubulin, a centriole marker in these spermatid steps (Fig. 7, Supplemental Figure S5).
TCFL5 protein expression and localization was not changed in the testes from Meig1 mutant mice
TCFL5 protein was examined in testis of Meig1 knockout mice. Western blot results indicated that there were no obvious differences in TCFL5 epitope abundance between wild-type and Meig1 mutant mice (Fig. 8). Immunofluorescence demonstrated that the protein was still present on the manchette of early elongating spermatids (Supplemental Figure S6) and centrioles of remaining late elongating spermatids (Supplemental Figure S7).
TCFL5 was identified in yeast two-hybrid screens using both MEIG1 and WD repeats of SPAG16 protein as bait. Given that both proteins are key regulators of spermatogenesis and spermiogenesis (Zhang et al., 2004, 2009), and MEIG1 associates with SPAG16 (Zhang et al., 2004), we hypothesized that TCFL5 would be found in the same complex, and also participate in the regulation of spermatogenesis. Given that both antibodies are generated in rabbits, this made it difficult to confirm the direct interaction in vivo using different immunological probes. Thus, the interaction between TCFL5 and MEIG1 was examined in transfected mammalian cells by co-immunoprecipitation experiment. To further study the gene, we characterized TCFL5′s tissue distribution, dynamic expression during the first wave of spermatogenesis and subcellular localization in vivo.
Mouse Tcfl5 message was present only in testis and was highly expressed at the onset of elongated spermatid formation (Kluin et al., 1982), which is consistent with the observation reported previously (Siep et al., 2004).
Mouse TCFL5 has conserved basic helix–loop–helix (bHLH) domain, characteristic of DNA-binding proteins and transcription factors. However, the fact that the protein was not present in the male germ cell nucleus suggests that it does not play a role in transcription. In addition, TCFL5 was expressed post-meiotically, when germ cell nuclei start condensing and gene transcription is beginning to shut down (Hogeveen & Sassone-Corsi, 2006; Pang et al., 2006).
The majority of proteins containing a bHLH are destined to enter the nucleus (Robbins et al., 1991); however, some proteins with this conserved domain do not enter. One example is SubH2Bv, a histone H2B variant (Aul & Oko, 2002). SubH2Bv has a bHLH domain, but in male germ cells the protein did not appear in the spermatid nucleus. Instead, it associated with proacrosomic and acrosomic vesicles, targeted to the nuclear surface to form the acrosome (Tran et al., 2011).
Another mouse TCFL5 was reported by (Siep et al., 2004). These authors identified the protein in a yeast one-hybrid screening using Calmegin gene promoter as bait. Northern blot and RT-PCR analyses showed that the message was expressed only in testis, and the message was significantly increased after 21 days of birth, which is consistent with our finding that the protein is highly expressed post-meiotically. Surprisingly, the authors generated two polyclonal antibodies using two peptides (labelled red in the Supplemental Figure S8), and discovered that the protein was present in late pachytene primary spermatocytes, and also in secondary spermatocytes that arise from primary spermatocytes through the first meiotic division, where the protein co-localized with transcriptionally active chromatin.
We hypothesize that the two TCFL5 proteins might be different isoforms translated from the same gene, which is supported by the fact that four TCFL5 isoforms were reported in the Ensembl database. When comparing the amino acid sequences of the two proteins, the first 48 amino acids of the TCFL5 identified by us were not present in the TCFL5 protein discovered by Siep (Supplemental Figure S8); thus, these initial amino acids might play a role in directing the protein localization in the manchette and centriole. In addition, the TCFL5 discovered by Siep had one extra amino acid (E273) that was not present in the TCFL5 protein identified by us, and W427 is C474 (Supplemental Figure S8). There was no explanation by Siep et al., 2004 as to why the Tcfl5 message was highly abundant post-meiotically, but the protein was expressed predominantly during meiosis. Given that the two antibodies generated by Siep and colleagues should recognize our TCFL5 protein, and our antibody clearly cross-reacts with the protein expressed post-meiotically, which matches the pattern of message expression, we cannot rule out the possibility that the two antibodies generated by Siep and colleagues were not specific against the TCLF5 protein.
The manchette is a transient microtubular structure assembled concurrently with the elongation and condensation of the spermatid nucleus and growth of the centrosome-derived axoneme. The appearance and disappearance of the manchette are likely related to the dynamic morphological changes in the spermatids during spermiogenesis (Clermont et al., 1993; Meistrich, 1993); thus, the timing of manchette development is very precise. It appears during early spermatid elongation and disappears when the elongation and condensation processes approach completion. Several findings suggest that the manchette can sort structural proteins to the centrosome and the developing sperm tail through a mechanism of intramanchette transport (IMT) (Kierszenbaum, 2001, 2002; Kierszenbaum & Tres, 2004). IMT and intraflagellar transport (IFT) share similar molecular components. IFT, initially discovered in flagella of Chlamydomonas (Rosenbaum & Witman, 2002), has been observed in mouse ciliated and flagellated cells. Mutations in IFT proteins disrupt ciliary biogenesis (Fan et al., 2010; Rix et al., 2011). Some IFT proteins, including IFT88 and IFT20, are also present in the manchette (Taulman et al., 2001; Sironen et al., 2010) and other cargo proteins have been identified, including ODF1, ODF2, SPAG4, SPAG5 (Burfeind & Hoyer-Fender, 1991; Schumacher et al., 1995; Brohmann et al., 1997; Shao et al., 1999, 2001). Dynein motor proteins, and some proteins that regulate dynein function and localization, including dynactin, lissencephaly 1, nuclear distribution protein E (NUDE) are also present in the manchette, some of which have been shown to be essential for spermatogenesis (Fouquet et al., 2000; Koizumi et al., 2003; Nayernia et al., 2003; Yan et al., 2003; Yamaguchi et al., 2004).
Our recent studies demonstrated that MEIG1 was localized to the manchette of elongating spermatids (Song J, Teves ME, Archer KJ, Tang W, Strauss JF, Zhang Z, unpublished observations). The interaction between TCFL5 and MEIG1 suggested that TCFL5 protein expression/localization might be altered in the Meig1-deficient mice. But the fact that TCFL5 was not affected in the Meig1-deficient mice indicated that TCFL5 may function upstream of MEIG1 protein.
The centriole is an organelle that is found at core of the mitotic spindle (Bornens, 2012). In dividing cells, the centriole moves to the cell surface during G1 where it functions as a basal body to template ciliogenesis. The central link between centrioles and cilia is highlighted by the fact that centrioles are only found in species that have cilia at some point in their life cycle. Basal bodies are specialized forms of centrioles, and basal body proteins can be classified into three categories. First, the core structural components, which include tubulin as well as other components of the centriole triplet microtubule blade, for example tektin (Hinchcliffe & Linck, 1998). The second class would be proteins that are recruited transiently to the basal bodies prior to their transport elsewhere, such as IFT and BBS proteins (Keller et al., 2005). The third class is the proteins that are permanently associated with the basal body, but that form associated fibrous structures, such as ODF2/cenexin (Kunimoto et al., 2012). Basal bodies are strictly required for formation of cilia. The key function of the basal body in ciliogenesis is most likely to provide the template for the formation of the axoneme (Piasecki & Silflow, 2009), basal body also functions in attaching and orienting cilia at cortex (Marshall, 2008). In addition, basal body also plays a role in regulating protein import into cilia (Kee et al., 2012). TCFL5 is highly concentrated in the centriole region of the late elongating spermatids strongly suggests that the protein might also play a role in sperm flagella/axoneme formation.
In conclusion, our studies demonstrated that the mouse TCFL5 57 kDa protein is not a nuclear protein in elongating spermatids. The localization of TCFL5 in the manchette and centriole suggests that it may play a role in spermiogenesis, possibly in the formation of the sperm flagellum in concert with MEIG1 and SPAG16.
This research was supported by Hubei Chutian Scholar Program, Virginia Commonwealth University Presidential Research Incentive Program (To ZZ) and National Natural Science Foundation of China (To JGF, project approval no. 81172462). M.E.T. is a visiting scholar from Centro de Biología Celular y Molecular, Universidad Nacional de Córdoba, Argentina, and supported by the Fogarty International Center (5D43TW007692).
We thank Dr. Jerome F. Strauss III (Virginia Commonwealth University) for his continuous support to our studies. Tissue processing and staining were performed in Histology Core Facility of Wuhan University of Science and Technology and Virginia Commonwealth University. Confocal microscopy was performed in South-Central University for Nationalities and the Imaging Core of Virginia Commonwealth University (5P30NS047463).